Efficient restricted gene expression in beta cells by lentivirus-mediated gene transfer into pancreatic stem/progenitor cells
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- Castaing, M., Guerci, A., Mallet, J. et al. Diabetologia (2005) 48: 709. doi:10.1007/s00125-005-1694-6
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Gene transfer into pancreatic beta cells, which produce and secrete insulin, is a promising strategy to protect such cells against autoimmune destruction and also to generate beta cells in mass, thereby providing a novel therapeutic approach to treat diabetic patients. Until recently, exogenous DNA has been directly transferred into mature beta cells with various levels of success. We investigated whether exogenous DNA could be stably transferred into pancreatic stem/progenitor cells, which would subsequently differentiate into mature beta cells expressing the transgene.
We designed transplantation and tissue culture procedures to obtain ex vivo models of pancreatic development. We next constructed recombinant lentiviruses expressing enhanced green fluorescent protein (eGFP) under the control of either the rat insulin promoter or a ubiquitous promoter, and performed viral infection of rat embryonic pancreatic tissue.
Embryonic pancreas infected with recombinant lentiviruses resulted in endocrine cell differentiation and restricted cell type expression of the transgene according to the specificity of the promoter used in the viral construct. We next demonstrated that the efficiency of infection could be further improved upon infection of embryonic pancreatic epithelia, followed by their in vitro culture, using conditions that favour endocrine cell differentiation. Under these conditions, endocrine stem/progenitor cells expressing neurogenin 3 are efficiently transduced by recombinant lentiviral vectors. Moreover, when eGFP was placed under the control of the insulin promoter, 70.4% of the developed beta cells were eGFP-expressing cells. All of the eGFP-positive cells were insulin-producing cells.
We have demonstrated that mature rat pancreatic beta cells can be stably modified by infecting pancreatic stem/progenitor cells that undergo endocrine differentiation.
KeywordsBeta cells Gene transfer ngn3 Recombinant lentivirus Stem/progenitor cells
enhanced green fluorescent protein
HEPES buffered saline solution
in situ hybridisation
rat insulin II gene promoter
severe combined immunodeficiency
vesicular stomatitis virus
Type 1 diabetes mellitus is due to an autoimmune process resulting in the destruction of pancreatic beta cells, the cells that produce and secrete insulin. Replacement of insulin-producing tissue by transplantation of mature beta cells represents an effective method of curing type 1 diabetes. However, many obstacles remain before cell replacement therapy  can be used routinely to treat diabetic patients . The major issues to be solved are allorejection, recurrence of anti-islet cell autoimmunity and the shortage of available donor tissue.
To address the immunological problems and increase the limited supply of human tissue available for grafts, attempts have been made to transfer exogenous DNA into mature beta cells. Several gene delivery viral systems, such as moloney-based retroviral vectors, adenoviral vectors, adeno-associated viral vectors, or lentiviral vectors have been employed for ex vivo gene transfer into pancreatic islet cells. Moloney-based retroviral vectors were inefficient in transducing mature insulin-expressing cells due to the lack of proliferation of mature beta cells . Adenoviruses were highly efficient in transducing non-dividing beta cells [3, 4, 5], however, the expression of the transgene was transient. This could be due either to the absence of genome integration of the virus or to the elimination of transduced cells by the host immune system . To circumvent this limitation in long-term expression, adeno-associated viral vectors have been used to transduce pancreatic islet cells [7, 8]. Lentiviral vectors were capable of transducing mature islet cells with sustained expression of either reporter genes [3, 9, 10], therapeutic molecules , or immortalising genes . Together, the above data indicate that exogenous DNA can be stably transferred to mature beta cells [3, 9, 10], providing limited protection against immune destruction . However, this approach failed to generate beta cell lines .
In the aforementioned studies, only mature beta cells were infected and transduced with various viral vectors. In cell types of the haematopoietic system, mature cells have been modified by viral infection of progenitor cells that undergo differentiation. Several groups have recently reported successful transduction of reporter genes or genes of therapeutical importance into progenitor cells that were taken from umbilical cord blood or bone marrow and differentiated into mature cells [12, 13, 14, 15]. In these experiments, the transgene was constitutively expressed, irrespective of the differentiation stage. More recently, Pawliuk and co-workers further modified the system using cell-type-specific promoters . They infected haematopoietic stem cells with recombinant lentiviruses expressing their gene of interest under the control of the beta globin locus control region, achieving high erythroid-specific gene expression levels .
While the results described above concern the haematopoietic system, only a limited number of studies in solid tissue show stable modification of mature cells by transduction of progenitor cells [17, 18, 19]. However, in these reports, only constitutive promoters were used, resulting in an expression of the transgene without cell type restriction.
Our objective was to determine whether beta cells can be modified by infecting stem/progenitor cells that will differentiate into beta cells. For this purpose, we designed ex vivo experimental models in which immature embryonic pancreases, rich in endocrine progenitors, develop into mature pancreatic tissue. We next analysed conditions to infect immature pancreatic tissues with recombinant lentiviruses expressing enhanced green fluorescent protein (eGFP) under the control of the insulin promoter. Our data demonstrate that recombinant lentiviruses can infect pancreatic stem/progenitors, such as cells positive for neurogenin 3 (ngn3) that will differentiate into beta cells expressing the transgene of interest. This strategy represents a new approach to the generation of genetically modified beta cells.
Materials and methods
DNA constructs and recombinant lentiviral production
The backbone of the lentiviral construct, pTRIP, has been previously described . The vector, pTRIP ΔU3.CMV-eGFP , expresses the eGFP gene under the control of an internal cytomegalovirus (CMV) promoter. A new lentiviral vector, pTRIP ΔU3.RIP405-eGFP, was constructed with the Rat insulin II gene promoter (RIP) in order to restrict the expression of eGFP to insulin-producing cells. A 405-bp fragment of RIP was amplified from the PBS-RIP-beta globin plasmid (kindly provided by P. Herrera, University of Geneva, Switzerland)  using the PCR expand system (Roche Diagnostic, Meylan, France) with the following primers: MluI RIP405 sense 5′cgacgcgtGGACACAGCTATCAGTGGGA3′ and BamHI RIP antisense 5′cgggatccTAGGGCTGGGGGTTACT3′.
The resulting PCR product was subcloned into the pGEMT-easy vector (Promega, Charbonnières, France) and the inserted RIP405 fragment was entirely sequenced. The MluI–BamHI digestion fragment was then inserted into a MluI–BamHI linearised promoterless pTRIP ΔU3.-eGFP vector (kindly provided by Dr A. P. Bemelmans, CNRS UMR 7091, Paris).
Lentiviral vector stocks were produced by transient transfection of 293T cells with the p8.7 encapsidation plasmid (ΔVprΔVifΔVpuΔNef) , pHCMV-G, encoding the vesicular stomatitis virus (VSV) glycoprotein-G  and the pTRIP ΔU3. recombinant vector as previously described . The supernatants were treated with DNAse I (Roche Diagnostic) prior to ultracentrifugation and the resulting pellet was resuspended in PBS, aliquotted and frozen at −80°C until use. The amount of p24 capsid protein was quantified by the HIV-1 p24 ELISA antigen assay (Beckman Coulter, Villepinte, France).
Promoter specificity in a lentiviral context: titrations and transduction efficiency
Preparation of pancreatic rudiments
Pregnant Wistar rats were obtained from Janvier (CERJ, Le Genest, France). All animal manipulations were performed according to the guidelines of the French Animal Care Committee. The morning post coitum was designated as embryonic day (E) 0.5. At E13.5 and E16.5 days of gestation, pregnant female rats were killed by CO2 asphyxiation. Rat embryos were removed from the uterus, the digestive tracts isolated and the pancreatic primordia dissected . Intact or partially dissociated embryonic pancreases were prepared for transplantation. The pancreatic buds were incubated with 0.16 mg/ml of Dispase I (Roche Diagnostic) for 20 min and subsequently mechanically dissociated. The purified pancreatic epithelium used for organotypical culture was separated from its surrounding mesenchyme by enzymatic and mechanical treatments as previously described [27, 28].
Infection of embryonic pancreases and transplantation
Recombinant lentiviruses were used to infect either intact or partially dissociated pancreases. Tissues were incubated with viral particles (120 ng of p24) for 2 h at 37°C with 5% CO2 atmosphere, in 200 μl of RPMI-1640 medium (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum containing HEPES (10 mmol/l), l-glutamine (2 mmol/l), non-essential amino acid (Invitrogen), penicillin (100 units/ml) and streptomycin (100 μg/ml). To increase the viral infection efficiency, diethylaminoethyl-dextran was added to a final concentration of 10 μg/ml. After lentiviral infection, tissues were washed twice with HEPES buffered saline solution (HBSS, Invitrogen) and kept on ice until transplantation into severe combined immunodeficient (scid) mice.
Seven-week-old, male scid mice (Charles River Laboratories, L’arbresle, France) were maintained in isolators.
Using a dissecting microscope, the pancreases were implanted under the kidney capsule as previously described , with the following modifications. The left kidney was exteriorised; a small transverse incision was made through the capsule on the ventral surface of the kidney, near the inferior pole. A small silicon cylinder was pushed under the capsule to provide a sealed space to confine the transplanted cells and tissues . The tissues were then introduced into the cylinder using forceps and/or a Hamilton syringe. At different time points after transplantation, the mice were killed, the kidney removed, and the graft dissected. Fixation with 3.7% formaldehyde was used prior to embedding in paraffin for immunohistological analysis. Consecutive 4-μm-thick sections were collected on gelatinised glass slides and immunofluorescent analysis was performed as described elsewhere [27, 31].
Infection of embryonic pancreases and organotypical culture
Embryonic pancreatic epithelia were infected as described above for whole pancreases. After a 2-h infection period, they were washed twice with HBSS and grown in three-dimensional collagen gels as described previously [27, 28]. Cultures were maintained for 1, 3 or 7 days. The rudiments were fixed in PBS containing 4% paraformaldehyde (Carlo-Erba Reactifs, Val de Reuil, France), cryoprotected in 15% sucrose–PBS at 4°C overnight, embedded in 15% sucrose–7% gelatin PBS and frozen at −50°C in isopentane (Carlo-Erba Reactifs). We collected 10-μm-thick cryosections and analysed them by immunohistochemistry or in situ hybridisation.
Immunofluorescent staining was performed according to the manufacturer’s instructions. Primary antisera, applied on paraffin sections, include mouse monoclonal anti-insulin or anti-glucagon (1/2,000; Sigma-Aldrich, Saint-Quentin Fallavier, France), anti-eGFP (1/400; BD Biosciences Clontech-Ozyme, Saint Quentin Yveline, France) and rabbit polyclonal anti-insulin or anti-glucagon (1/2,000; DiaSorin, Anthony, France), anti-pan-cytokeratin (1/500; Dako, Trappes, France), and anti-carboxypeptidase A (1/600; Biogenesis/Valbiotech AbCys, Paris, France), antibodies. The fluorescent secondary antibodies obtained from Jackson Immunoresearch Laboratories (Beckman Coulter) were fluorescein anti-mouse antibodies (1/150), fluorescein anti-rabbit antibodies (1/200) and Texas-red anti-mouse or rabbit antibodies (1/400). Photographs of the sections were taken using a fluorescence microscope (Leica, Leitz DMRB, Rueil-Malmaison, France) and digitised using a Hamamatsu (Middlesex, NJ, USA) C5810 cooled 3CCD camera.
For immunofluorescence detection on cryosections, primary antibodies used were mouse monoclonal anti-eGFP (1/400; Abcam, Cambridge, UK) and rabbit polyclonal anti-insulin (1/2,000; DiaSorin). The species-specific affinity-purified secondary antibodies included FITC-conjugated goat anti-mouse (1/150; Caltag Tebu, Burlingame, CA, USA) and CY3-conjugated goat anti-rabbit IgG (1/400; Jackson ImmunoResearch Laboratories).
Single-labelled sections incubated with mismatched secondary antibodies showed no immunostaining, confirming the specificity of the secondary antisera. Photographs of the sections were taken on a Leica, Leitz TCS confocal microscope with a krypton–argon laser beam.
Tissue sections were stained with bis benzimide Hoechst 33258 trihydrochloride (Sigma-Aldrich). The quantitative analysis of the number of GFP-positive and/or insulin-expressing cells per epithelium after culture was performed by counting the number of immunoreactive cells for which a nucleus was clearly detected.
Cold in situ hybridisation
Cold in situ hybridisation (ISH) was performed as described previously [32, 33]. Antisense riboprobes were generated from cDNA or PCR fragments subcloned into the pGEMTeasy vector (Promega). The entire coding region of the eGFP fragment (711 bp) was amplified from the pTRIP ΔU3.-eGFP plasmid with the following primers: 5′-ATGGTGAGCAAGGGCGAG-3′ and 5′-TACTTGTACAGCTCGTCC-3′. Both antisense and sense ngn3 riboprobes (726 bp) were prepared as previously described . Plasmids were linearised and used as templates for the synthesis of sense or antisense riboprobes by T7 or SP6 RNA polymerase (Promega), in the presence of digoxygenin-UTP (Roche Diagnostic). For double in situ hybridisation, either digoxygenin-UTP (for ngn3) or fluorescein-UTP (for eGFP) was used. Colorimetric revelations were performed with 5-bromo-4-chloro-3-indolyl phosphate and either nitroblue tetrazolium (Promega) for digoxygenin-UTP, or 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-phenyl-tetrazolium chloride (Roche Diagnostic) for fluorescein-UTP, to obtain the blue or red staining, respectively.
Cold in situ hybridisation and immunofluorescence double labelling
ISH was performed with an ngn3 digoxygenin-labelled antisense riboprobe and revealed in blue as described above. After several washes in PBS, eGFP immunofluorescence labelling was achieved according to the manufacturer’s instructions with a rabbit polyclonal anti-eGFP antibody (1/400; BD Biosciences Clontech-Ozyme) and a fluorescein anti-rabbit secondary antibody (1/200; Jackson Immunoresearch Laboratories).
Specificity of the rat insulin promoter in a lentiviral context in vitro
Development of rat embryonic pancreases engrafted in mice
The increase in beta cell mass during the transplantation period could be due either to the proliferation of the rare existing insulin-expressing cells, or to the differentiation of progenitor cells into mature beta cells. To discern between the two possibilities, we thus repeated the transplantation experiments with E13.5 pancreases, which do not contain beta cells at this stage (Fig. 2e), whereas, glucagon-expressing cells (Fig. 2e) or CPA-containing cells (Fig. 2g) are barely present. Transplantation of these pancreases was performed as previously described and analysed 7 days later (Fig. 2f, h). Exocrine tissue developed as revealed by the detection of CPA-expressing cells with normal acinar morphology (Fig. 2h). In addition, we observed insulin- and glucagon-expressing cells forming structures resembling islets of Langerhans (Fig. 2f). These results clearly indicate that the increase of the beta cell mass observed in our model is mainly due to the differentiation and not the proliferation of pre-existing beta cells.
Together, these data demonstrate that we generated a functional ex vivo model for rat pancreatic development. When engrafted under the kidney capsule of scid mice, E13.5 and E16.5 immature pancreases develop into endocrine and exocrine tissues with normal morphology.
Transduction with recombinant lentiviruses mediates long-term expression of cell-type-specific eGFP expression in insulin-positive cells
In our in vivo model, we sought to determine whether recombinant lentiviruses can infect progenitor cells that will differentiate into beta cells specifically expressing the transgene. E13.5 and E16.5 pancreases were infected with the pTRIP ΔU3.RIP405-eGFP vector, grafted and analysed 7 days later. Under these conditions, no eGFP-expressing cells were detected (data not shown).
To define the cell types expressing eGFP in the infected pancreases that developed in the scid mice, double immuno-labelling was performed using antibodies directed against insulin (beta cells), glucagon (alpha cells), pan-cytokeratin (duct cells), or carboxypeptidase-A (acinar cells) in combination with anti-eGFP. As shown in Fig. 3a, 7 days after transplantation eGFP was clearly detected in insulin-expressing cells, while eGFP was never detected in glucagon-expressing cells (Fig. 3b), pan-cytokeratin-expressing cells (Fig. 3c) or CPA-expressing cells (Fig. 3d). We also analysed the grafts 1 month after transplantation. As shown in Fig. 3e, sustained beta cell expression of eGFP was achieved.
Highly efficient lentiviral transduction of immature pancreatic rudiments
As described above, the pTRIP ΔU3.RIP405-eGFP vector transduces E13.5 and E16.5 pancreatic rudiments, with eGFP transgene expression being restricted to insulin-producing cells. However, the transduction efficiency was less than 5% of the insulin-containing cells.
The explants were infected in parallel with viruses where eGFP transcription was under the control of the CMV promoter and cultured for 3 or 7 days. As shown in Fig. 5c and d, a large number of eGFP-expressing cells that stained either positive or negative for insulin were observed, reflecting the ubiquitous expression feature of the CMV promoter. In addition, the percentage of eGFP-positive cells relative to the total number of cells was not significantly different after 3 or 7 days in culture (33.8 and 41.3%, respectively; Fig. 6b, right panel). This demonstrates that in our organotypical culture model, the ubiquitous CMV promoter drives the expression of a transgene in a manner independent of the differentiation status.
Beta cells expressing eGFP derive from infected progenitors
In this study, we have demonstrated that recombinant lentiviral vectors can efficiently infect pancreatic progenitor cells and thereby stably modify mature rat pancreatic beta cells.
During the past years, different approaches have been used to genetically modify beta cells with two major objectives: beta cell protection against immune destruction; and beta cell expansion. For these purposes, recombinant DNA was transferred into mature beta cells using different vectors, such as adenoviruses, adeno-associated viruses and lentiviruses . While progress has been made in ways of protecting beta cells against immune destruction , attempts to expand beta cells by gene transfer directly into insulin-producing cells have been unsuccessful. For example, beta cell lines generated by infection with retroviral vectors expressing the SV40 large T antigen, H-RAS and hTERT oncogenes lose insulin expression with time, suggesting that the cells de-differentiate . Beta cell differentiation could be reactivated by transfection of specific transcription factors combined with the use of specific growth factors. However, even in such conditions, the levels of insulin expression remained low when compared to mature beta cells . Other investigators were unable to stably immortalise beta cells using lentiviruses expressing the SV40 T antigen . Although gene transfer was efficient, the inability to obtain an immortalised cell line may be due to the use of mature beta cells as infected target cells. To circumvent this limitation, we have addressed here the use of lentiviral vectors as a tool to stably transduce pancreatic endocrine progenitor cells, instead of mature beta cells.
We designed an ex vivo transplantation experimental model in which progenitor cells differentiate into beta cells. We grafted embryonic pancreases under the kidney capsule of immuno-incompetent mice. This grafting site has been shown to permit the development of a large number of tissues, such as rodent thymus, bovine adrenocortical cells and human embryonic and fetal pancreases [29, 30, 40, 41]. Within 1 week after transplantation of E16.5 pancreases, the insulin content was multiplied by a factor of 250. Such a beta cell increase could not be due to the proliferation of the few pre-existing insulin-expressing cells for the following reasons. First, beta cell proliferation is very limited during prenatal development . Second, it is now clearly established that the rare insulin-expressing cells present during development of the embryonic pancreas and which frequently co-express glucagon are not the progenitors of mature beta cells [42, 43]. Lastly, E13.5 pancreases that do not contain beta cells develop into beta cells when grafted to scid mice (the present work). Thus, our data indicate that the observed development of endocrine tissue in our ex vivo experimental model is due to the differentiation of progenitor cells originally present in the transplanted tissue.
In order to infect the pancreatic progenitor cells and subsequently transduce mature beta cells, access of the virus to the progenitor cell population is a crucial experimental step. There are different arguments indicating that pancreatic progenitor cells are located in the centre of the pancreatic tissue: (1) during embryonic life, the cells expressing ngn3, a marker of endocrine pancreatic progenitor cells, are located in the central region of the developing pancreas that corresponds to the pancreatic epithelium [36, 44, 45, 46]; (2) the first insulin-expressing cells also appear in the central region of the pancreas . However, it is well established that in compact tissues, the accessibility of viral particles to cells located in the centre of the tissue is poor . We show here that by partially dissociating pancreases, we increase the probability of contact between the virus and the progenitor cells and demonstrate that progenitor cells that differentiate into beta cells can indeed be infected. The infection yield of progenitors was further enhanced by direct exposure of purified embryonic pancreatic epithelium to the viruses upon removal of the surrounding mesenchyme.
As discussed above, our data indicate that the beta cells expressing eGFP derived from the infection of progenitor cells that differentiate into beta cells rather than from the infection of pre-existing beta cells that proliferate. It was, therefore, of crucial importance to characterise the nature of the cells that were the primary cell target infected by the lentiviral vectors. We showed that numerous ngn3-positive cells expressed eGFP 24 h after infection with a lentiviral vector, in which eGFP expression was under the control of the ubiquitous CMV promoter. This demonstrates that endocrine progenitor cells can be infected and a restricted transgene expression obtained according to the specificity of the promoter.
The experimental process described here represents a powerful tool for gain-of-function experiments in a dynamic developmental model that can allow promoter-specific targeting throughout the entire cell differentiation cascade in the pancreas. In addition, our model is also appropriate for loss-of-function studies, since recent data have shown that RNA interference can be efficiently performed using lentiviral vectors [48, 49, 50].
We designed, in this study, a new model that resembles those used in vivo to produce rodent beta cell lines. In these cases, insulinomas were produced from transgenic mice expressing an oncogene under the control of the insulin promoter allowing the expression of the transgene specifically in mature beta cells [24, 51]. This approach was used to generate rodent beta cell lines that were functional in terms of insulin secretion upon glucose simulation. However, the beta cell lines were obtained by gene transfer in fertilised eggs, which restricts its application to animal models without any possible transfer to humans. The model presented here is based on ex vivo gene transfer, which is applicable to both animal and human tissues. Recently, we designed an ex vivo system, where human embryonic pancreases can develop into mature pancreatic tissues . Such a model will now be used to test the feasibility of infecting human pancreatic progenitor cells with a specific expression of the transgene in mature human beta cells.
The first two authors contributed equally to this work. This work was supported by the Juvenile Diabetes Research Foundation (JDRF Center for Beta Cell Therapy in Europe), INSERM/Fondation pour la Recherche Médicale/Juvenile Diabetes Research Foundation (4DA03H), the Association Française des Diabétiques (AFD), the Fondation pour la Recherche Médicale, INSERM, the region Ile de France, Retina France and the University Paris VI.
The authors are deeply grateful to Yasmine Hazhouz, Emilie Neveu, Isabelle LeNin and Stephanie Bauchet for technical assistance and to Medeva Ghee and Chamsy Sarkis for helpful comments and critical reading of the manuscript. Muriel Castaing was successively supported by the Foundation pour la Recherche Medicale (FRM) and by l’Association d’Aide aux Jeunes Diabétiques (AJD). Aline Guerci was successively supported by the Ministère de la Recherche et de la Technologie and by the FRM.