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. 2014 Apr 2;90(5):109. doi: 10.1095/biolreprod.113.115600

Sustained Expression of Insulin by a Genetically Engineered Sertoli Cell Line after Allotransplantation in Diabetic BALB/c Mice1

Gurvinder Kaur 3, Lea Ann Thompson 3, Mithun Pasham 3, Kim Tessanne 4, Charles R Long 4, Jannette M Dufour 3,2,
PMCID: PMC4076370  PMID: 24695630

ABSTRACT

Immune-privileged Sertoli cells (SCs) exhibit long-term survival after allotransplantation or xenotransplantation, suggesting they can be used as a vehicle for cell-based gene therapy. Previously, we demonstrated that SCs engineered to secrete insulin by using an adenoviral vector normalized blood glucose levels in diabetic mice. However, the expression of insulin was transient, and the use of immunocompromised mice did not address the question of whether SCs can stably express insulin in immunocompetent animals. Thus, the objective of the current study was to use a lentiviral vector to achieve stable expression of insulin in SCs and test the ability of these cells to survive after allotransplantation. A mouse SC line transduced with a recombinant lentiviral vector containing furin-modified human proinsulin cDNA (MSC-EhI-Zs) maintained stable insulin expression in vitro. Allotransplantation of MSC-EhI-Zs cells into diabetic BALB/c mice demonstrated 88% and 75% graft survival rates at 20 and 50 days post-transplantation, respectively. Transplanted MSC-EhI-Zs cells continued to produce insulin mRNA throughout the study (i.e., 50 days); however, insulin protein was detected only in patches of cells within the grafts. Consistent with low insulin protein detection, there was no significant change in blood glucose levels in the transplant recipients. Nevertheless, MSC-EhI-Zs cells isolated from the grafts continued to express insulin protein in culture. Collectively, this demonstrates that MSC-EhI-Zs cells stably expressed insulin and survived allotransplantation without immunosuppression. This further strengthens the use of SCs as targets for cell-based gene therapy for the treatment of numerous chronic diseases, especially those that require basal protein expression.

Keywords: cell-based gene therapy, immune privilege, insulin, lentivirus, mouse Sertoli cell line, Sertoli cells, testis, transplantation

A mouse Sertoli cell line (MSC-1) genetically engineered using a lentiviral construct carrying furin-modified human proinsulin cDNA stably expresses insulin mRNA and protein and survives allotransplantation into diabetic BALB/c mice.

INTRODUCTION

Gene therapy has the potential to greatly improve medical treatment, as it can replace faulty or absent genes. Direct gene delivery through viral vectors is hampered by immune response generated against the viral vector and insertional mutagenesis [1]. Cell-based gene therapy, defined as the use of cells engineered to carry a protein of interest to target specific diseases, is an effective way to overcome most of the issues of viral vector-based gene delivery [2]. However, cells carrying the transgene will still elicit an immune response from the host and thus require the use of immunosuppressive therapy.

Sertoli cells (SCs) located within the seminiferous epithelium of the testis provide structural and nutritional support to developing germ cells [3, 4]. Because most germ cells develop after the immune system is fully functional, they are autoimmunogenic. Besides nurturing these autoimmunogenic germ cells, SCs also protect them from an immunologic response by expressing and secreting several immunoregulatory factors, thus contributing to testis' immune privilege. Immune modulation by SCs is not restricted to the testis as SCs also survive when transplanted to ectopic sites (outside the testis), across immunological barriers (i.e., allo- or xenotransplantation), without the need for immunosuppressive drugs [5–13]. This suggests that immune-privileged SCs used as a vehicle for cell-based gene therapy have the potential to overcome the obstacle of immune rejection.

To test whether genetically engineered SCs retain their immune-privileged properties, SCs expressing green fluorescent protein (GFP) were transplanted as allografts underneath the kidney capsule of naïve BALB/c mice. Transplanted SCs survived and continued to express GFP throughout the study (at least 60 days) [14]. Thereafter, the ability of SCs to express and secrete therapeutic proteins was explored. SCs transduced with a recombinant adenoviral vector, carrying human neurotropin-3 (hNT-3) produced biologically relevant levels of hNT-3 in vitro. However, due to the transient nature of the adenoviral vector, the function of hNT-3 in vivo was not demonstrated, as these cells stopped expressing the transgene 3 days after transplantation [15]. Recently, prepubertal mouse and porcine SCs transduced with an adenoviral vector containing furin-modified human proinsulin cDNA restored normoglycemia after transplantation in diabetic SCID mice [16]. Nonetheless, the expression levels of insulin and normoglycemia were transient due to the use of adenovirus and proliferating prepubertal SCs. Furthermore, that study used immunocompromised animals as transplant recipients [16], and consequently, the question of whether genetically engineered SCs can stably express the transgene in immunocompetent mice remained.

When short-lived, high expression of a transgene is required, recombinant adenoviral vectors are very efficient, but due to their epichromosomal nature, they do not stably integrate into the cells' DNA, and expression of the transgene can be lost [17]. Induction of host immune response against adenoviral antigens further hinders long-term transgene expression [18]. Lentiviral vectors' innate ability to integrate into the host genome makes them an ideal vector to achieve long-term expression of the transgene [19], and as most of the genes encoding viral proteins have been removed, they are less immunogenic than adenoviral vectors [20].

The objective of the current study was to use a lentiviral vector to achieve stable expression of insulin in MSC-1 cells. MSC-1 cells are derived from a mouse Sertoli cell line isolated from a testicular tumor collected from the testes of C57Bl/6 X SJL mice carrying a transgene containing simian virus (SV) 40 large T antigen fused to the transcriptional regulatory sequence of human Müllerian inhibiting substance [21]. MSC-1 cells have been shown to maintain numerous characteristics of primary SCs [22], including survival when transplanted as allografts in diabetic mice [23]. Moreover, their immortality makes them ideal for testing the stability of the lentiviral construct. In this study, MSC-1 cells were transduced with a lentiviral vector containing furin-modified human proinsulin cDNA, and cells were analyzed for stable expression of insulin mRNA and protein. Transduced cells were then transplanted as allografts into diabetic mice with a fully functional immune system, and their survival and ability to express insulin were measured.

MATERIALS AND METHODS

Animals

Male BALB/c mice (Charles Rivers Laboratories) 8–9 wk of age were used as transplant recipients. Mice were rendered diabetic by an intraperitoneal injection of streptozotocin (270–275 mg/kg body weight; Sigma Chemical Co.) 1 wk prior to transplantation. Only those animals exhibiting nonfasting blood glucose values >20 mM were used as recipients. After transplantation, blood samples obtained from the tail veins of nonfasted recipients were tested by glucose assay once a week (OneTouch Ultra; LifeScan). All animals were maintained under appropriate conditions in accordance with Institute for Laboratory Animal Research Care and Use of Laboratory Animals and Texas Tech University Health Sciences Center Institutional Animal Care and Use Committee-approved protocols and guidelines of the National Institutes of Health.

Production and Transduction of Recombinant Lentivirus

Recombinant lentivirus carrying furin-modified human proinsulin cDNA and ZsGFP under the control of elongation factor 1α promoter (EhI-Zs [Fig. 1]) was produced, and virus titers were determined as described previously [24]. MSC-1 cells were cultured in Dulbecco modified Eagle medium (DMEM; Sigma-Aldrich) at 37°C and transduced overnight with the EhI-Zs lentiviral particles (2U per 1 μl of polybrene). The next day, fresh medium (DMEM containing 5% fetal bovine serum [FBS; v/v]) was added to the cells. After 60 h, cells were selected for 1 wk by using 1000 μg/ml G418, and stably transduced cells were maintained in DMEM plus 10% FBS containing 250 μg/ml G418 throughout the study. Controls included MSC-1 cells that were not transduced and cultured in DMEM plus 10% FBS without G418.

FIG. 1.

FIG. 1

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Lentiviral plasmid EhI-Zs. The plasmid contains elongation factor 1α promoter (EF1α), which drives the expression of furin-modified human proinsulin cDNA (hINS) and ZsGreen fluorescent protein (ZsGreen). For selection in bacterial and mammalian cells, the plasmid contains the neomycin (Neo) and zeocin (Zeo) antibiotic resistance genes. RRE, rev response element; IRES, internal ribosome entry site; LTR, long terminal repeat.

RNA Extraction and RT-PCR

Transduced MSC-1 cells (MSC-EhI-Zs), nontransduced MSC-1 cells, and grafts collected at Days 20 and 50 post-transplantation were lysed in 1 ml of TRIzol reagent (Invitrogen), and total RNA was extracted according to the manufacturer's protocol. The RNA was DNase-treated (Roche) and used as template for oligo(dT)-primed cDNA synthesis, using Multiscribe reverse transcriptase (Applied Biosystems) and TaqMan reverse transcriptase reagent (Applied Biosystems). PCR was performed for insulin and the housekeeping gene cyclophilin, using Fast Start PCR Master Mix (Roche). Insulin and cyclophilin were amplified from 50 ng of cDNA under the following PCR conditions: 94°C for 2 min; then 35 cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec; and a final extension of 72°C for 10 min. PCR fragments were separated on a 1% TAE (1× TAE = 40 mM Tris-acetate, 1 mM EDTA) agarose gel at 90V for 45 min, and fragments were visualized by ethidium bromide staining. All amplicons obtained were of the expected size (263 bp for insulin and 458 bp for cyclophilin). Primers used were human insulin forward, 5′-GGG ACC TGA CCC AGC CGC A, and reverse, 5′-CAG GCT GCC TGC ACC AGG G, and cyclophilin, forward 5′- CCC ACC GTG TTC TTC GAG, and reverse, 5′-ATC TTC TTG CTG GTC TTG CC.

Immunocytochemistry for Insulin and GFP

MSC-EhI-Zs or MSC-1 cells (1 × 105 cells/well) were cultured on chamber slides in DMEM containing 10% FBS with or without 250 μg/ml G418, respectively. After 24 h, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Slides were blocked with 20% normal goat serum (v/v) and incubated with guinea pig polyclonal anti-swine insulin (1:1000 dilution; DAKO), mouse monoclonal anti-GFP (1:100 dilution; Millipore) or mouse monoclonal anti-SV40 large T antigen (1:100 dilution; BD Biosciences) primary antibodies as described previously [14, 16, 23]. After primary antibody incubation, slides were incubated with Alexa Fluor 594 goat anti-guinea pig (1:400 dilution; Life Technologies) or Alexa Fluor 488 goat anti-mouse (1:400 dilution; Life Technologies) secondary antibodies. Slides were then incubated with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI; 1 μg/ml; Molecular Probes, Inc.) to detect cell nuclei. Negative controls were put through the same procedure without primary antibody. All negative controls lacked a positive reaction. Images were acquired by microscopy (Axio Star plus microscope, using AxioCam MRc digital camera, using Axio vision Rel 4.8 software; Carl Zeiss) and were digitally combined into figures (Photoshop version 7.0 software; Adobe).

Human Insulin ELISA

MSC-EhI-Zs or MSC-1 cells (2.5 × 105 cells/well) were cultured on chamber slides in DMEM containing 10% FBS with or without 250 μg/ml G418, respectively. After 48 h, medium was collected and stored at −80°C until analyzed. Quantification of the amount of human insulin secreted was performed using human insulin ELISA kit (Linco Research, Inc., St. Charles, MO) as described previously [16]. DMEM was used as the negative control.

Transplantation and Graft Characterization

Prior to transplantation, MSC-EhI-Zs or MSC-1 cells were cultured on nontissue-culture-treated Petri dishes containing Ham F10 medium with supplements [16, 23] and 10% FBS for 48 h at 37°C to allow formation of aggregates. Six million MSC-EhI-Zs or MSC-1 cells, calculated using a double-stranded DNA quantitation assay (PicoGreen) [18], were transplanted into the left renal subcapsular space of isoflurane-anesthetized diabetic BALB/c mice, as described previously [16, 23, 25]. Graft-bearing kidneys were collected at Days 20 and 50 post-transplantation for RT-PCR, immunohistochemistry, or cell culture.

For immunostaining, graft-bearing kidneys were immersed in Z-fix (Anatech LTD), embedded in paraffin, and tissue sections were stained for insulin, GFP, and SV40 large T antigen (MSC-1 cell marker) as described previously [14, 16, 23]. Primary antibodies included guinea pig polyclonal anti-swine insulin (1:100 dilution; DAKO), mouse monoclonal anti-GFP (1:25 dilution; Millipore), or mouse monoclonal anti-SV40 large T antigen (1:100 dilution; BD Biosciences). Sections were then incubated with the appropriate biotinylated secondary antibodies (1:200 dilution; Vector Laboratories) followed by incubation with ABC-enzyme complex (Vector Laboratories). Diaminobenzidine (DAB; Biogenex) was used as chromogen, and sections were counterstained with hematoxylin. Negative controls were put through the same procedure without primary antibody. All negative controls lacked a positive reaction. Images were acquired by microscopy as described previously.

To obtain single-cell suspensions for cell culture, grafts were carefully removed from the kidney, and tissue was chopped gently in Hanks balanced salt solution (Sigma-Aldrich). The tissue was incubated with 1 mg/ml trypsin (Roche) and 0.4 mg/ml DNase (Roche) at 37°C for 10 min. The dissociated cells were then cultured on chamber slides in DMEM containing 10% FBS with or without 250 μg/ml G418. After 2 days, cells were fixed with 4% paraformaldehyde, and immunofluorescence was performed as described above (Immunocytochemistry for Insulin and GFP).

RESULTS

Long-Term Production of Insulin by MSC-EhI-Zs Cells

MSC-1 cells were transduced with a lentiviral vector carrying furin-modified human proinsulin cDNA and ZsGreen fluorescent protein downstream of elongation factor 1α promoter (MSC-EhI-Zs [Fig. 1]). These transduced MSC-EhI-Zs cells were maintained for over 2 yr (through multiple freeze-thaw cycles) and analyzed at various times for insulin and GFP expression. MSC-EhI-Zs cells continued to express insulin mRNA for at least 2 yr (Fig. 2A, lane 2), whereas insulin mRNA was never detected in nontransduced MSC-1 cells (Fig. 2A, lane 4). MSC-EhI-Zs cells also continued to express GFP (Fig. 2B, green) and insulin (Fig. 2C, red) proteins, whereas GFP and insulin protein were not detected in nontransduced MSC-1 cells (Fig. 2, D and E). The amount of insulin secreted from MSC-EhI-Zs or MSC-1 cells into the cell culture medium was analyzed by human insulin ELISA. MSC-EhI-Zs cells secreted 1 × 10−8 μg/cell. Insulin secretion by nontransduced MSC-1 cells was below the detection limit of the ELISA kit. Thus, transduction of MSC-1 cells with EhI-Zs lentiviral particles led to stable integration of the furin-modified human proinsulin cDNA construct into MSC-1 cells as demonstrated by long-term insulin expression and secretion by these cells for at least 2 yr. However, the amount of insulin secreted by these cells was approximately 100-fold less than the amount secreted by primary mouse or porcine Sertoli cells transduced with an adenoviral vector, as reported in our previous study [16].

FIG. 2.

FIG. 2

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Production of insulin mRNA and protein by MSC-EhI-Zs or MSC-1 cells. MSC-EhI-Zs and MSC-1 cells were cultured in DMEM plus 10% FBS with and without 250 μg/ml of G418, respectively. After 2 days, cells were either collected for RNA isolation or fixed for immunofluorescence. A) RT-PCR was performed for insulin (lanes 2 and 4) and cyclophilin (lanes 3 and 5), using RNA isolated from MSC-EhI-Zs (lanes 2 and 3) or MSC-1 (lanes 4 and 5) cells. Lane 1 is the 1-kb Plus DNA Ladder (Invitrogen). Immunofluorescence was performed to detect GFP (green [B and D]) and insulin (red [C and E]) protein in MSC-EhI-Zs (B and C) or MSC-1 (D and E) cells. Cells were counterstained with DAPI to detect cell nuclei (blue [B–E]).

Survival and Stable Insulin Production by MSC-EhI-Zs Cells after Allotransplantation

Six million MSC-EhI-Zs or MSC-1 cells were transplanted under the kidney capsule of diabetic BALB/c mice as allografts, and blood glucose levels, cell survival, and insulin expression were measured. Due to the low amount of insulin secretion by the MSC-EhI-Zs cells, the transplanted cells had no effect on blood glucose levels and did not restore normoglycemia in the transplanted diabetic mice (data not shown). Nevertheless, the MSC-EhI-Zs cells survived and continued to express insulin (Table 1). When the allografts were collected at Day 20, 88% of MSC-EhI-Zs and 86% of MSC-1 cell grafts contained SV40 large T antigen-positive cells (Table 1). At Day 50 post-transplantation, 75% of the MSC-EhI-Zs and 70% of the MSC-1 cell grafts were positive for SV40 large T antigen (Table 1). Most of the cells within the grafts were surviving MSC-EhI-Zs or MSC-1 cells (Fig. 3), indicating that most of the recipient mice did not reject the transplanted allografts. Moreover, RT-PCR performed using RNA isolated from the MSC-EhI-Zs cell grafts collected at both 20 and 50 days after transplantation was positive for insulin mRNA (Table 1; Fig. 4A, lanes 2 and 3). Consistent with the low secretion of insulin by the MSC-EhI-Zs cells, only a few patches of cells in MSC-EhI-Zs cell grafts were positive for GFP and insulin proteins at 20 and 50 days post-transplantation (Table 1; Fig. 4, B and C). Nontransduced MSC-1 cell grafts lacked insulin mRNA (Fig. 4A, lanes 4 and 5). Furthermore, insulin protein and GFP were not detected in the nontransduced MSC-1 cell grafts (Table 1; Fig. 4, D and E).

TABLE 1.

Cell survival and long-term production of insulin and GFP by MSC-EhI-Zs or MSC-1 cells.

graphic file with name i0006-3363-90-5-109-t01.jpg

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a 

Survival was determined by SV40 large T antigen immunostaining.

b 

ND, not determined.

FIG. 3.

FIG. 3

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Analysis of MSC-EhI-Zs or MSC-1 cell allografts for cell survival. Six million MSC-EhI-Zs (A and B) or MSC-1 (C and D) cells were transplanted as allografts into diabetic mice, and graft-bearing kidneys were collected at 20 (A and C) and 50 (B and D) days post-transplantation. Tissue sections were immunostained for SV40 large T antigen (brown [A–D]) as a marker for MSC-1 cells. The graft and kidney are separated by a dotted line. All sections were counterstained with hematoxylin to detect cell nuclei. Insets are higher magnifications (50 μm) of A–D. K, kidney.

FIG. 4.

FIG. 4

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Insulin and GFP expression in MSC-EhI-Zs or MSC-1 cell grafts. A) RT-PCR was performed for insulin, using mRNA isolated from MSC-EhI-Zs (lanes 2 and 3) or MSC-1 (lanes 4 and 5) cell grafts collected at 20 (lanes 2 and 4) and 50 (lanes 3 and 5) days post-transplantation. Lane 1 is the 1-kb Plus DNA Ladder (Invitrogen). MSC-EhI-Zs (B and C) or MSC-1 (D and E) cell graft-bearing kidneys were collected at Day 50 post-transplantation and immunostained for GFP (brown [B and D]) and insulin (brown [C and E]). The graft and kidney are separated by a dotted line. All sections were counterstained with hematoxylin (blue [B–E]). Insets are higher magnifications (50 μm) of B–E. Arrows in B and C indicate GFP or insulin-positive cells, respectively. K, kidney.

Insulin Protein Expression by Cells Collected from the Grafts

Detection of only a few GFP and insulin positive MSC-EhI-Zs cells in the grafts could have been due to the low level of protein expression, which was further masked by the tissue paraffin embedding and processing procedures. To determine whether the transplanted cells within the grafts were still expressing GFP and insulin, MSC-EhI-Zs or MSC-1 cell grafts were collected at Days 20 and 50 post-transplantation and dissociated into single cells. The isolated cells were cultured in vitro and analyzed for MSC-1 cell markers and for insulin and GFP expression. Most of the cells isolated from MSC-EhI-Zs or MSC-1 cell grafts were positive for SV40 large T antigen at both 20 and 50 days post-transplantation (Table 1 and Fig. 5, A and C, red), indicating that the in vitro cultured cells were MSC-1 cells. Analysis of the cells isolated from the MSC-EhI-Zs or MSC-1 cell grafts for insulin and GFP expression revealed that most of the MSC-EhI-Zs cells were still expressing insulin protein and GFP at Days 20 (Table 1; Figs. 5B and 6, A and B) and 50 (Table 1 and Fig. 6, C and D) post-transplantation, suggesting that the tissue processing procedure could be responsible for masking detection of the insulin protein and GFP in vivo. Insulin protein and GFP expression levels were not detected in the cells isolated from nontransduced MSC-1 cell grafts (Table 1 and Fig. 5D).

FIG. 5.

FIG. 5

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SV40 large T antigen expression in cells isolated from MSC-EhI-Zs or MSC-1 cell allografts. Cells were isolated from MSC-EhI-Zs (A and B) or MSC-1 (C and D) cells graft collected at Day 50 post-transplantation and cultured in vitro in DMEM plus 10% FBS with or without 250 μg/ml G418, respectively. The cultured cells were immunostained for SV40 large T antigen (red [A and C]) and GFP (green [B and D]). All sections were counterstained with DAPI (blue [A and B]).

FIG. 6.

FIG. 6

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Insulin and GFP expression in cells isolated from MSC-EhI-Zs cell grafts. Cells isolated from MSC-EhI-Zs cell grafts collected at Days 20 (A and B) and 50 (C and D) post-transplantation were immunostained for GFP (green [A and C]) and insulin (red [B and D]). All sections were counterstained with DAPI to detect nuclei (blue [A–D]).

DISCUSSION

Viral vector-based gene therapy has potential to deliver genes of interest for treatment of disease. However, insertional mutagenesis and generation of host immune response against viral vectors raise concerns related to the safety of this therapy and hamper its use in clinical trials [1, 26, 27]. Due to the epichromosomal nature of adenoviral vectors, insertional mutagenesis is not a concern; however, this can prevent stable expression of the transgene. Adenoviral vectors also retain most of their viral genome and thus induce strong innate and cell-mediated immune responses [26]. Lentiviral vectors, which incorporate genetic material into the chromosome, are less immunogenic than adenoviral and retroviral vectors, although it has been shown that an immune response can be elicited against lentivirus-encoded transgene products and the vector itself [28–32]. Several in vivo animal studies have also shown that these vectors can exhibit insertional mutagenesis [31, 33].

Cell-based gene therapy is an alternative that avoids viral vector-based insertional mutagenesis. For instance, cells genetically engineered to carry the transgene can be expanded in culture and screened for mutagenesis, and the cells that express high levels of the transgene and are devoid of vector insertion near proto-oncogenes could be used for clinical transplantation (to replace the faulty or defective gene). The major limitation of this therapy is that the individual must take immunosuppressive drugs throughout his or her life to avoid an immune response from the host that results in the rejection of the transplanted cells carrying the transgene. The ability of immune-privileged Sertoli cells to survive across immunological barriers (i.e., allo- or xenotransplantation in rodents and humans without immunosuppressive drugs [5, 7, 12, 25]) make them an ideal vehicle for cell-based gene therapy.

Viral (adenovirus, retrovirus, and lentivirus) vectors have been used successfully to transduce Sertoli cells [34–37]. For instance, in vivo transduction of pubertal “nondividing” Sertoli cells with an adenoviral vector carrying the full-length mouse Steel gene partially restored spermatogenesis in infertile Steel/Steeldickie (Sl/Sld) mutant mouse testes at 6–10 wk after virus injection. These animals remained infertile, but round spermatids or spermatozoa isolated from the testes of these animals resulted in offspring after microinsemination [35]. In a separate study, the transduction efficiency of adenoviral vector, immunological response generated against the transgene (Lac Z) and its effect on spermatogenesis were determined [38]. Although the adenoviral vector led to strong transgene expression, the testes from the adenovirus-injected mice showed a deleterious effect on spermatogenesis, as evident by decreased spermatocytes and increase in apoptotic index [38]. Furthermore, adenovirus transduction induced an inflammatory immune response composed of CD4 and CD8 T-cell infiltration and significant increase in interleukin 6 (IL6) and IL8 secretion in testes of the transduced animals compared to that in controls [38]. The use of lentiviral vector for in vivo transduction of Sertoli cells with functional c-kit ligand (KL2) in Sl/Sld mouse testes led to the stable expression of the transgene (more than 5 mo) in Sertoli cells and restored spermatogenesis in all recipient testes without deleterious effects. Moreover, spermatid and spermatozoa isolated from transduced testes were able to produce normal offspring after intracytoplasmic sperm injection [34].

Initial exploration of the use of Sertoli cells as vehicles for cell-based gene therapy demonstrated that Sertoli cells can be genetically engineered to express foreign proteins (e.g., GFP and hNT-3) [14, 15]. However, those studies did not demonstrate in vivo function of the transgene. In a more recent study, we examined whether Sertoli cells could be genetically engineered to express and secrete insulin by transducing prepubertal Sertoli cells with adenoviral vector carrying furin-modified human proinsulin cDNA [16]. Transplantation of these genetically engineered Sertoli cells lowered blood glucose levels in diabetic SCID (immunocompromised) mice [16]. However, due to the epichromosomal nature of adenoviral vectors and proliferating nature of prepubertal Sertoli cells, the decrease in blood glucose levels was transient, and animals returned to the diabetic state within 8 days [16]. This study demonstrated that Sertoli cells engineered to express a therapeutically relevant protein (insulin) are capable of expressing the functional gene product at levels adequate for the treatment of disease (diabetes mellitus), even if for a short period of time. However, in order to strengthen the utility of Sertoli cells as a novel tool for cell-based gene therapy to treat a chronic disease, the next major step was to create a vector that allowed stable in vivo expression of the transgene by Sertoli cells and demonstrated that these cells (stably expressing insulin) could escape host immune response without immunosuppressive drugs. To achieve that goal, a mouse Sertoli cell line was transduced with lentiviral particles carrying furin-modified human proinsulin cDNA (MSC-EhI-Zs). Lentiviral transduction led to the stable expression of insulin by MSC-EhI-Zs cells as these cells retained the insulin mRNA and protein expression after multiple freeze-thaw cycles for at least 2 yr. However, insulin protein secretion by MSC-EhI-Zs cells was low compared to that in Sertoli cells transduced with an adenoviral vector (1 × 10−8 μg/cell vs 1.5 × 10−6 μg/cell, respectively), which could be due to the low transduction efficiency of lentiviral vectors. For adenoviral vectors, multiple copies of the virus are delivered to the cell, whereas only 1–2 copies of the lentiviral genome (carrying transgene of interest) are integrated into the cell [39, 40]. Nevertheless, MSC-EhI-Zs cells transplanted as allografts survived and produced insulin mRNA throughout the study (i.e., Day 50 post-transplantation), although, GFP and insulin proteins were detected in only a few of the cells within the sectioned grafts. Detection of low levels of insulin- and GFP-positive cells in vivo could be explained by low protein levels that were further masked by the tissue processing technique, as most of the MSC-EhI-Zs cells expressed insulin and GFP in vitro prior to transplantation. Additionally, most of the MSC-EhI-Zs cells isolated from the grafts and cultured in vitro were positive for GFP and insulin at Days 20 and 50 post-transplantation. Due to low insulin production, transplanted MSC-EhI-Zs cells did not restore normoglycemia in the diabetic mice.

Overall, we were able to demonstrate stable production of insulin by MSC-1 cells and survival of these cells after allotransplantation in diabetic mice with an intact immune system. Although expression levels at this time were not high enough to treat diabetes, this therapy has great possibilities to treat disease where high expression of the transgene is not required (e.g., hemophilia [factor VIII or IX] and spinal cord injury [neurotropins]). In order to utilize this approach to treat type 1 diabetes, future studies are ongoing to generate a vector that will provide high insulin expression and secretion that will normalize blood glucose levels long-term.

Footnotes

1

Supported in part by National Institutes of Health grant HD067400 to J.M.D. from the Eunice Kennedy Shriver National Institute of Child Health and Human Development. Presented in part at the Joint International Congress of the Cell Transplant Society/International Xenotransplantation Association, October 23–26, 2011, Miami, Florida.

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