Endocrine replacement through islet transplantation is able to restore normal metabolic control in patients with type 1 diabetes; however, long-term islet allograft survival remains elusive due to immune rejection. Moreover, the current requirement for lifelong immunosuppression, as well as donor shortage, limits the widespread implementation of such a therapy (1, 2).
Sertoli cells, which reside within the seminiferous tubules of the testis (3), are believed to be responsible, at least in part, for the immune privileged environment of the testis by producing factors that act locally to inhibit immune response. Fas ligand (FasL) (4), transforming growth factor β (TGF-β) (5, 6), and clusterin (7), are potent immune modulators found in Sertoli cells that have been postulated to be involved in testicular immune privilege.
It has been previously demonstrated that rodent Sertoli cells can survive as allografts (4, 8) and that islet allografts are protected from immune-mediated rejection when cotransplanted with allogenic Sertoli cells without systemic immunosuppression (9). Because the clinical application of allogenic Sertoli cells is impractical due to donor shortage, using xenogenic porcine Sertoli cells may solve this problem. It has been recently reported that Neonatal porcine Sertoli cells (NPSCs) achieve long-term survival following xenotransplantation in non-immunosuppressed rats (10). However, whether cotransplantation of NPSCs with islet allografts can prolong survival in rats is still unknown.
In this study, we tested survival of cultured NPSCs in non-immunosuppressed Wistar rats when transplanted underneath the kidney capsule. Transplants were then performed to determine if islet allografts from Sprague-Dawley (SD) rats placed under the renal subcapsular space of diabetic Wistar rats could be immunoprotected when cotransplanted with NPSCs. We found that xenogenic NPSCs were capable of prolonging survival of allogeneic islets in a dose-dependent manner.
MATERIALS AND METHODS
Animals
Male large white neonatal pigs (aged 10–15 days, Huazhong university of Agriculture, Wuhan, China) were used as Sertoli cell donors. Female SD rats (aged 8–10 weeks, Tongji medical college, Wuhan, China), were used as islet cell donors. Female Wistar rats (aged 6–8 weeks, Disease Control and Prevention center, Hubei province, China), were used as transplant recipients. All recipients were rendered diabetic with alloxan (Sigma, USA) at a dose of 200 mg/kg by intraperitoneal injection 14 to 21 days before transplantation. Only those animals exhibiting nonfasting blood glucose values more than or equal to 22 mmol/L were used as recipients.
Cell Isolation and Culture
The NPSCs were isolated using a technique similar to that we previously described for rat Sertoli cells (11). Briefly, 10 to 15 day-old male pigs were intramuscular anesthetized with ketamine (1.5 mg/kg), testicles were aseptically removed and placed in Petri dishes containing 4°C DMEM/F-12 medium. The testes were cut into 1-mm pieces with scissors and digested for 10 to 15 min at 37°C with 0.2% (wt/vol) collagenase type V (Sigma, USA) and then washed with Hank's balanced salt solution (HBSS) supplemented with 0.02% (wt/vol) ethylenediamine-tetraacetic acid and 0.5% (wt/vol) bovine serum albumin. The tissues were resuspended in calcium- and magnesium-free HBSS and further digested with 0.25% (wt/vol) trypsin and 0.5 mg/mL DNase I (Sigma, USA) for 20 to 30 min at 37°C. The digest was passed through a 200-μm stainless mesh, and then washed with HBSS. The final pellets were suspended with DMEM/F-12 medium with 5% fetal calf serum. The isolated cells were cultured for 3 days in cell culture flasks (50 cm2) with a density of 4 to 5×106 at 37°C in 5% CO2 atmosphere before testing for FasL and Sox9 in vitro and use in transplantation experiments.
As the method described previously (12), rat pancreatic islets were prepared by collagenase type V digestion, Ficoll density gradient purification, and cultured for 1 day at 37°C in 5% CO2 atmosphere in RMPI 1640 medium supplemented with 20% fetal calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin.
Sox9 and FasL Expression of NPSCs In Vitro
As a specific marker of NPSCs, Sox9 was detected by reverse-transcription polymerase chain reaction (RT-PCR). Briefly, total RNA was extracted from NPSCs using Trizol reagent (Life Technologies, Rockville, MD). RNA yield and purity was determined spectrophotometrically at 260 to 280 nm. After DNase I digestion (Gibco, Life Technologies, Basel, Switzerland), 3 μg of total RNA was reverse-transcribed using the Revert AidTM First Strand cDNA Synthesis Kit (MBI cat. no. K1621) with oligo (dT)18 primer in a total reaction volume of 10 μL. Primers were designed as follows: sox9, the forward primer was 5′-CACTGGGAACAGCCCGTCTA-3′, the reverse primer was 5′-AAGGTGGTAATGCGTTTGG-3′, and the product length was 242bp. β-actin, the forward primer was 5′-GTGCGGGACATCAAGGAGAA-3′, the reverse primer was 5′-TGTCCACGTCGCACTTCAT-3′, and the product length was 241bp. β-actin was amplified as the internal control to allow semiquantitative evaluation of the expression levels. After reverse transcription, the following thermal profile was performed: 30 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, and final extension of 72°C for 5 min. Subsequently, RT-PCR products were resolved in 1.2% agarose gels stained with ethidium bromide. The gels were scanned, and the band intensities were evaluated.
Sox9 expression was further confirmed by immunocytochemistry. Briefly, freshly isolated cells were cultured at 37°C in 5%CO2 atmosphere in poly-L-lysine-coated coverslips for 12 hr to permit adherence. Cells were fixed with 95% alcohol for 30 min, washed by distilled water, blocked with H2O2 and methanol for 30 min to avoid endogenous peroxidase, and then added in 5%BSA for 20 min. Thereafter, 24 hr incubation was performed with rabbit anti-human sox9 antibody (1:100 dilutions, Santa Cruz, USA). Goat anti-rabbit IgG were used as secondary antibody. The negatively controls were performed by using nonrelated antibodies.
To determine whether cultured NPSCs expressed FasL, total RNA was extracted from NPSCs as described earlier. Total RNAs from the testis tissues of 14- or 60-day-old male pigs were used as controls. Primers were designed as follows: FasL, forward, 5′-TTTCAGCTCTTCCACCTAC-3′, reverse, 5′-CATCTTTCCCTCCATCAG-3′, and the product length is 384 bp.
Survival of NPSCs in Rats
To assess the survival of NPSCs as xenografts in vivo, 1.5×106 of NPSCs alone were implanted under the left renal capsule of each female Wistar rat in the absence of immunosuppression. The xenograft-bearing kidneys were harvested under sterile conditions at 3, 7, 14, 21, and 40 days posttransplant (n=3 for each time point).
Tissue from grafts and non-graft-bearing kidneys were immediately frozen and stored at −80°C for later RNA extraction. RNA isolation and RT-PCR of Sox9 were described earlier. The rat β-actin was amplified as the internal control, the forward primer was 5′-CACCAACTGGGACGATATG-3′, the reverse primer was 5′-AGGCATACAGGGACAACAC-3′, and the product length is 206 bp. Additionally, the graft-bearing kidneys were further examined with immunohistochemistry for Sox9. The tissues were immersed in formalin overnight, embedded in paraffin, and sections were incubated with rabbit anti-human sox9 antibody (1:100 dilutions, Santa Cruz, USA, reported [13]) at 37°C for 60 min. Sections were then incubated with goat anti-rabbit secondary antibody (1:100 dilutions, DAKO, USA) for 30 min followed by peroxidase-streptavidin, diaminobenzadine and then counterstained with hematoxylin. Negative controls were omission of primary antibody.
Cotransplantation of NPSCs and Allogeneic Islets in Rats
Each diabetic recipient received 1500 hand-picked SD islets (≥150 μm diameter) alone (as the control group, n=8) or in combination with two different doses of NPSCs (1.5×106, n=8 or 1.0×107, n=8). Composite grafts were prepared by mixing islets and NPSCs aggregates in polypropylene micro centrifuge tubes. Grafts were aspirated into a 1-mL syringe, centrifuged, and then gently injected to the left renal subcapsular space of anesthetized recipient rats.
To investigate whether NPSCs could provide immunoprotection to islet allografts implanted at a different site of the same recipient, 1500 IEQ SD islets were implanted under the left renal capsule and 1.0×107 of NPSCs were transplanted under the right renal capsule of the same recipient rat (n=4).
Recipient rats were allowed to recover, and blood glucose was assayed every day for the first week after transplantation and then three times per week afterwards. Graft-bearing kidneys (n=3) were harvested at the time of rejection (return to hyperglycemia, two consecutive readings of ≥11.2 mmol/L) or at 7 days posttransplant (n=2).
Pathologic and Immunohistochemical Analysis
The graft-bearing kidneys were immersed in formalin overnight, embedded in paraffin, and 5-μm thick sections were stained with hematoxylin-eosin. For immunohistochemistry, sections were immunostained with mouse anti-rat insulin (1:50 dilutions, Boster, Wuhan, China) or rabbit anti-human sox9 antibody (1:100 dilutions, Santa Cruz, USA), respectively. The slides were then incubated with goat anti-mouse or rabbit second antibody (1:100 dilutions, DAKO, USA).
Immunohistochemical Staining for Bcl-2 and HO-1
Immunohistochemical staining for Bcl-2 was performed on 5-μm formalin-fixed and paraffin-embedded sections. Briefly, after deparaffinization and rehydration, slides followed antigen retrieval in an autoclave (10 min at mild heat). Subsequently, they were incubated in methanol 3% H2O2 for 10 min at room temperature to inactivate endogenous perosidase. Slides were preincubated with 10% normal goat serum for 30 min at 37°C to block nonspecific binding. Tissue sections were then incubated with mouse anti-rat Bcl-2 antibody (Santa Cruz, sc-7382, CA, USA) diluted at 1:50 overnight at 4°C. After PBS washing, visualization was performed using the EnVision Detection Systems Peroxidase/DAB, Rabbit/Mouse (DAKO, K5007, Denmark). Slides were subsequently counterstained with hematoxylin, dehydrated in alcohol and xylene, and cover slipped with permanent mounting media.
Immunohistochemical staining for HO-1 was also performed using 5 μm sections of paraffin-embedded tissues. After the similar procedures as mentioned earlier, tissue sections were then incubated with rabbit anti-rat HO-1 antibody (Stressgen, SPA-895, Ann Arbor, USA) at 1:1000 overnight at 4°C. The slides were then incubated with Envision/HRP, Rabbit/Mouse (DAKO, K5007, Denmark) for 20 min at room temperature. And then visualization was performed using the 3, 5-diaminobenzidine. Slides were counterstained with hematoxylin, dehydrated, and mounted with neutral resin.
Statistical Analysis
All data were expressed as mean±standard deviation. The statistical significance of difference between multiple comparisons was calculated by one-way analysis of variance (ANOVA). P value less than 0.05 was considered significant.
RESULTS
Characterization of NPSCs
An enriched fraction of NPSCs was obtained by our modified digestion method. After cultured at 37°C in 5% CO2 atmosphere, NPSCs represent more than 90% of the total cell isolated; the viability of Sertoli cells as detected by Trypan blue staining was more than 95%. Sox9 mRNA was expressed on the NPSCs as detected by RT-PCR (Fig. 1B). Additionally, expression of Sox9 was positive on the NPSCs membrane and cytoplasm with immunocytochemistry staining (Fig. 1A–a).
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FIGURE 1.:
Sox9 and FasL expression in neonatal porcine Sertoli cells (NPSCs). (A) Immunocytochemical staining of NPSCs with anti-Sox9 antibody (a); stained with secondary antibody only was used as negative control (b). (B) Reverse-transcription polymerase chain reaction result of Sox9 mRNA expression in NPSCs isolated from 14-day old porcine testis. (C) Reverse-transcription polymerase chain reaction result of FasL expression in NPSCs isolated from 14-day old porcine testis (lane 1), 14-day old porcine testicular tissues (lane 2), and 60-day-old porcine testicular tissues (lane 3). A representative sample of three independent experiments is shown.
To evaluate the immune tolerogenicity of the cultured NPSCs, we detected the expression of FasL that is thought to confer Sertoli cells immunological privilege. Interestingly, the mRNA expression in the NPSCs was almost negative with the semiquantitative RT-PCR (Fig. 1C, lane 1); however, the mRNA expression of FasL in testiclular tissues from 14-day old pigs was positive, with levels of expression was further increased in testicles from 60-day-old pigs (Fig. 1C, lanes 2 and 3).
Survival of NPSCs in Nonimmunosuppressed Rats
To determine whether cultured NPSCs could survive as xenografts in vivo, 1.5×106 NPSCs were implanted under the kidney capsule of immunocompetent Wistar rats. At 3, 7, 14, 21, and 40 days posttransplant, graft-bearing kidneys were assessed by immunopathology and RT-PCR. Macroscopically, Sertoli cell grafts were easily observed at 21 days posttransplant, whereas they were relatively difficult to detect at 40 days posttransplant (Fig. 2A–a, b). When tissue sections were examined by immunohistochemistry, the Sox9 positive NPSCs were predominantly arranged as clusters of cells at 21 days posttransplant (Fig. 2A–c, e), however, they were significantly reduced at 40 days posttransplant (Fig. 2A–d, f).
FIGURE 2.:
Survival of neonatal porcine Sertoli cells (NPSCs) in non-immunosuppressed rats. (A) Identification of NPSCs grafts underneath the renal capsule of Wistar rats. Grafts were removed at 21 days (a), 40 days (b) posttransplant and photographed for macroscopic identification of transplanted tissue. Immunohistochemical staining for Sox9 at 21 (c, ×200; e, ×400), 40 (d, ×200; f, ×400) days posttransplant. (B) Detection of Sox9 mRNA in NPSCs graft-bearing kidneys. Grafts were removed at 3 days (lane1), 7 days (lane 2), 14 (lane 3), 21 (lane 4), 40 (lane 5) days posttransplant. Nontransplanted Wistar rat kidney tissues were used as negative controls (lane 6).
To further confirm the survival of NPSCs under the rat renal capsule, RT-PCR for porcine Sox9 gene was performed. Rat kidney tissues transplanted in absence of NPSCs were used as negative controls. As shown in Figure 2 (B), Sox9 was detected at the expected size of 242 bp in grafts at 3, 7, 14, 21 days posttransplant (lanes 1–4), however, it was nearly negative at 40 days posttransplant (lane 5). These data indicated that NPSCs could survive at least 21 days under the renal capsule in Wistar rats and are gradually destroyed afterwards.
The Effect of NPSCs on Islet Allograft Survival
To study whether xenogeneic NPSCs could prolong islet allograft survival, 1500 IEQ SD islets were transplanted alone or in combination with 1.5×106 or 1.0×107 NPSCs to diabetic female Wistar rats. All recipients of islet allografts alone achieved euglycemia within 2 days, but returned to a diabetic state by 4 to 7 days posttransplantation (mean survival time, 5.67±0.94 days; Figs. 3A and 4). When 1500 IEQ SD islets were cotransplanted with 1.5×106 NPSCs to the same site of the recipient, all animals again normalized within 2 days, but returned to hyperglycemia by 8 to 9 days posttransplantation (mean survival time, 8.33±0.58 days; Figs. 3B and 4). However, when the quantity of cotransplanted NPSCs was increased to 1.0×107, all of the animals remained normoglycemic until 15 to 18 days posttransplantation (mean survival time, 16.33±1.53 days; P<0.01 vs. islets alone group; Figs. 3C and 4). Interestingly, when 1500 IEQ SD islets and 1.0×107 NPSCs were transplanted separately to the different site of the recipient, all animals achieved euglycemia within 2 days, but quickly returned to a diabetic state by 5 to 6 days posttransplantation (mean survival time, 5.25±0.5 days; P>0.05 vs. islets alone control group; Fig. 3D).
FIGURE 3.:
Function of islet allografts transplanted into diabetic Wistar rats. Nonfasting blood glucose concentrations of the diabetic Wistar rats after islets transplanted alone (A); cotransplanted with 1.5×106 neonatal porcine Sertoli cells (NPSCs) at the same site (B); cotransplanted with 1.0×107 NPSCs at the same site (C); or cotransplanted with 1.0×107 NPSCs at different side of kidneys (D).
FIGURE 4.:
Survival curve of islet allograft in different groups. Cotransplanted with 1.0×107 Neonatal porcine Sertoli cells significantly prolonged the islet allograft survival (P<0.01, vs. the other 2 groups).
Histologic Examination of NPSCs Xenografts and Islet Allografts
Histologic analysis of graft-bearing kidneys was performed by hematoxylin-eosin and immunohistochemical staining to detect the presence of pancreatic β-cells (insulin) and cotransplanted NPSCs (Sox9) (Fig. 5). Seven days posttransplant, graft tissue harvested from the control animals revealed the presence of few insulin-positive cells, which were surrounded by extensive lymphocytic infiltrates (Fig. 5A, B). However, compared with the control grafts, the composite grafts from animals receiving 1.0×107 NPSCs (harvested at the 7 days posttransplant) demonstrated the presence of more insulin- positive cells compared with control grafts. Additionally, considerably fewer lymphocytic infiltration was observed (Fig. 5C, D). At the same time, immunohistochemical staining confirmed the existence of extensive Sox9-positive porcine Sertoli cells (Fig. 5E, F). In contrast, at the time of rejection (18 days posttransplant), graft tissue harvested from the 1.0×107 NPSCs groups revealed the presence of less insulin-positive cells, which were surrounded by infiltrating lymphocytes (Fig. 5G, H). Moreover, amount of Sox9-positive porcine Sertoli cells were also significantly reduced (Fig. 5I, J), which may associated with the rejection of islet allografts.
FIGURE 5.:
Immunohistochemical analysis of islet allografts and composite neonatal porcine Sertoli cells (NPSCs) xenografts. At 7 days posttransplant, immunohistochemical staining for insulin of the islet allografts in the control group, (A, ×200; B, ×400) and the 1.0×107 NPSCs group (C, ×200; D, ×400). At 18 days posttransplant, staining for insulin of the islet allografts in the 1.0×107 NPSCs group (G, ×200; H, ×400). Additionally, sox9 was immunohistochemical stained to detect the cotransplanted NPSCs in the 1.0×107 NPSCs group at 7 days (E, ×200; F, ×400) and 18 days (I, ×200; J, ×400) posttransplant.
Expression of Immunoprotective Genes
Immunohistochemical staining for Bcl-2 and HO-1 was performed on the graft-bearing renal tissues. NPSCs transplanted alone did not express Bcl-2 (at day 21, Fig. 6A), whereas islets transplanted alone only showed weak expression of Bcl-2 at the implanted sites (at day 7, Fig. 6B). However, when NPSCs were cotransplanted with islets to the same site, Bcl-2 expression was significantly upregulated in the subcapsular of the rat kidney (at day 18, Fig. 6C), indicating that NPSCs may promote the local expression of Bcl-2. NPSCs transplanted alone or islets transplanted alone or cotransplantation of both had similar positive HO-1 expression at the implanted sites (Fig. 6D, F).
FIGURE 6.:
Immunohistochemical analysis of Bcl-2 (A–C, ×200) and HO-1 (D–F, ×200) using graft-bearing renal tissues in neonatal porcine Sertoli cells transplanted alone group (at 21 days posttransplant, A, D) or islets transplanted alone control group (at 7 days posttransplant, B, E) or the group of cotransplantation with 1.0×107 neonatal porcine Sertoli cells and islets at the same site (at 18 days posttransplant, C, F).
DISCUSSION
Recently, it has been reported that transplantation of co-cultured neonatal porcine islets and Sertoli cells, inserted into a preimplanted perforated metal cylinder, resulted in insulin independence for more than 1 year in type 1 diabetic patients without the use of immunosuppression (14). This suggests the possibility of cotransplanting xenogenic Sertoli cells to enhance viability of cellular grafts, perhaps through locally inhibiting the immune response of recipients. However, when NPSCs are transplanted with porcine islets without use of the closed perforated metal cylinder, prolongation of islet survival did not occur in rats or in nonhuman primates (15, 16). Thus, while conflicting results exist regarding potential of xenogenic Seroli cells to protect xenogenic islets. To our knowledge, the question of whether cotransplanted xenogenic Sertoli cells (NPSCs) can protect allogeneic islet allografts has not been addressed.
To investigate such possible immunoprotective effect on cotransplanted cellular grafts, it is important to know first whether NPSCs themselves can survive long term as xenografts in nonimmunosuppressed recipients. Dufour et al. transplanted 11×106 cultured NPSCs under the kidney capsule of immune competent Lewis rats and demonstrated long-term survival (at least 90 days) of NPSC xenografts by vimentin immunostaining and PCR for the porcine mitochondrial cytochrome oxidase II subunit gene. Sox9 is a member of the Sox family of transcription factors containing a SRY-related HMG box and is responsible for maintaining the Sertoli cell phenotype and testis development. Expression of Sox9 is evolutionarily conserved and is specific for Sertoli cells in pig and other species (17–19). As recently reported (13), Sox9 has been shown to be a specific marker for Sertoli transplantation studies. Therefore, survival of NPSCs was determined by porcine Sox9 RT-PCR and further verified by Sox9 immunostaining in this study. We demonstrated that NPSCs could survive more than 21 days, but less than 40 days, when 1.5×106 of cultured NPSCs were transplanted underneath the kidney capsule of each non-immunosuppressed Wistar rat. One possible reason for differences between this study and that of Dufour et al. (10) is the lower concentration of NPSCs we used (1.5×106) compared with their study (11×106). Although Sox9 is evolutionarily conserved and specific for Sertoli cells, the possibility may exist that the transplanted NPSCs were losing the capacity/stimuli to produce Sox9 rather than the loss of these cells. However, it seems that the prolonged survival of NPSCs as xenografts may have the ability to protect the cotransplanted islet allografts.
In this study, we demonstrated that cotransplantation with sufficient xenogenic NPSCs could significantly prolong islet allograft survival in non-immunosuppressed diabetic rats. Pathology and immunopathology showed the presence of more insulin-positive cells and considerably fewer lymphocytes infiltration in the composite grafts from animals receiving 1.0×107 NPSCs than the control grafts at 7 days posttransplant. Moreover, immunopathology also revealed the presence of abundant NPSCs in cotransplantation group, which were positive for Sox9, near insulin-positive cells. These results indicate that cotransplanted NPSCs might protect islet allografts through inhibition of local lymphocytes infiltration. To our knowledge, the present study is the first to report that xenogenic NPSCs can provide immunoprotection and prolong the survival of islet allografts in rats without any immunosuppression or immune-modulating intervention. Furthermore, 1.0×107 of NPSCs implanted in the right renal subcapsular space could not prolong the survival of islet allografts placed in the contralateral kidney, indicating that NPSCs mainly provided local immunoprotection to cotransplanted islet allografts and not a systemic tolerogenic effect.
Although xenogenic NPSCs showed in vivo immunoprotective effects on both themselves and cotransplanted islet allografts in rats, the precise mechanisms are still unclear. Previously, data indicated that FasL expressed by Sertoli cells may play a role in the survival of mouse testicular tissue fragments transplanted under the kidney capsule of allogeneic recipients (4). In particular, testicular tissue isolated from gld mice (lacking functional FasL) transplanted as allografts were no longer present after 7 days while grafts from wild type mice (producing functional FasL) survived for 28 days (4). However, recent studies by others indicated a paradoxical role of FasL in immune privilege (20, 21). Specifically, it has been reported that transgenic expression of FasL on islet cells causes a more rapid rejection of islets accompanied by granulocyte or neutrophilic infiltration (22–24). In our study, NPSCs from 10 to 15 day old pigs were weak, if at all, positive for FasL, indicating that FasL may not be the central contributing factor to the observed immunoprotective effect of NPSCs. Other studies have shown that the cytokine TGF-β is required for immune privilege (25, 26) and infiltrating precursor CTLs fail to acquire direct cytotoxic function in the privileged sites (27). When using the NOD mouse Sertoli cell/islet cotransplant model, TGF-β was demonstrated to play a protective role in preventing islet destruction (6). Whether TGF-β is involved in the immunoprotection of our models needs to be further investigated. In this study, the expression of some immunoprotective genes was detected by immunohistochemistry. We observed that NPSCs seem to promote the localized expression of Bcl-2 at the implantation site, which might contribute to the immunoprotective effects provided by NPSCs.
In conclusion, cotransplantation with NPSCs could provide local immunoprotection and significantly prolong islet allograft survival in non-immunosuppressive rats, which might have potential in future clinical islet allotransplantation.
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