The introduction of the Edmonton Protocol in 2000 confirmed that islet transplantation can provide a population of patients with T1DM excellent glycemic control and insulin independence (1). Expansion of clinical islet transplantation has been limited by the large requirement for donor tissue, since at least two islet transplant procedures (combined mass of >10,000 Islet Equivalents (IEQ)/kg body weight) are typically required to establish insulin independence in recipients, which presents a considerable obstacle given the prevalence of diabetes and the limited supply of suitable cadaveric donor organs (1–3).
In order to broaden the availability of islet transplantation to the majority of patients with T1DM, alternative sources of insulin-producing tissue must be identified. Pigs are particularly attractive as a xenogeneic islet donor since they are widely available, produce insulin that is functional in humans, and could be selected for certain donor characteristics. However, despite many studies aimed at optimizing adult pig islet isolation, no protocols have been established that generate consistent, reproducible islet yields (4–6). Also, following the isolation, adult pig islets are notoriously fragile in the tissue culture setting, limiting the possibility to pool multiple donor preparations or manipulate the islets in vitro prior to transplantation (4–7). Experimentation over the last decade using neonatal porcine islets (NPI) has suggested that the difficulties associated with adult pig islets could be avoided, since high yields of NPI can be easily and reproducibly isolated and maintained in culture for several weeks (8). Even though NPI are immature and contain less β-cell mass than adult islets, they are responsive to glucose challenge in vitro and can establish euglycemia in diabetic, immunodeficient animals following a 6–8 week in vivo maturation period (8). Recent data have shown that NPI allografts transplanted intraportally into allogeneic adult pigs or xenogeneic macaques reverse hyperglycemia in approximately one month (9, 10). Thus, NPI show considerable promise as a potential source of donor tissue in clinical islet transplantation.
The current clinical need for large numbers of donor islets is directly related to the fact that most of the transplanted tissue fails to survive within the recipient. β-cells are known to be especially sensitive to hypoxia and to chemokine/cytokine-mediated damage, and these pro-apoptotic stresses are present both during isolation and during the early posttransplant period. Using murine models of syngeneic islet transplantation, it has been demonstrated that even under ideal circumstances (i.e. absence of any immune rejection response), >60% of cells within islet grafts are lost due to apoptosis (11). In clinical islet transplantation it has been suggested that at least two thirds of the implanted islets never become functional (2, 11, 12). The impact of this early posttransplant graft loss is immediately evident because more islets must be implanted to achieve insulin independence.
Studies using mature rodent and human islets have demonstrated that the widespread apoptotic cell death occurring within islets during the early posttransplant period can be attributed to several factors related to oxidative stress. The process of islet isolation and purification causes the rapid onset of hypoxia (13). After infusion into the recipient portal circulation, the postisolation hypoxic period (pO2 of 5–10 mmHg or <1% O2) is extended for up to two weeks, until revascularization occurs, which in turn leads to reoxygenation injury and further apoptosis in islets (14–17). The sensitivity of NPI to the stresses occurring in the early posttransplant period is currently unknown. Because NPI represent a potential source of tissue for clinical islet transplantation, and since posttransplant apoptosis is a key determinant of islet mass required to treat diabetes (and potentially of long-term antigraft immunity), in the present study the sensitivity of NPI to hypoxia, hypoxia-reoxygenation, and posttransplant apoptosis was investigated.
MATERIALS AND METHODS
βTc-Tet refers to a pancreatic β-cell line derived from an insulinoma of transgenic C3H mice; these mice express tetracycline-regulated SV40 large T antigen specifically within pancreatic β-cells (18, 19). βTc-Tet cells were maintained as described (20). The cells were allowed to proliferate continuously in all studies.
Landrace-Yorkshire neonatal pigs were obtained from the University of Alberta Farm (Edmonton, AB). Immunodeficient NOD-RAG−/− mice (NOD.129S7(B6)-Rag1tm1Mom/J) were obtained from the Jackson Laboratory (Bar Harbor, ME) and housed under specific pathogen-free conditions. Ethical approval was obtained from the animal welfare committee at the University of Alberta, and all animals were cared for according to the guidelines of the Canadian Council on Animal Care.
NPI were isolated from 1–3 day old Landrace-Yorkshire neonatal pigs (1.5–2.0 kg) using the method developed by Korbutt et al. (8). The pancreas was removed from anesthetized animals following laparotomy and exsanguination and placed into Hanks' balanced salt solution (Invitrogen Canada). The pancreas was cut into small pieces, digested with 2.5 mg/ml collagenase (Sigma-Aldrich Canada, Mississauga, Ontario), and filtered through a 500 μm nylon mesh. The filtrate containing NPI was cultured for 7–10 days at 37 oC, 20% CO2 (ordinary culture conditions) in Ham's F10 medium supplemented with 180 mg/dL glucose, 50 μM isolbutalmethylxanthine (IBMX, ICN Biomedicals, Montreal, PQ), 0.5% bovine serum albumin (BSA, fraction V, radioimmunoassay grade, Sigma Aldrich Canada), 2 mM l-glutamine, 3 mM CaCl2, 10 mM nicotinamide, 100 U/ml penicillin, and 100 μg/ml streptomycin (the latter 5 reagents all from Invitrogen Canada, Burlington, Ontario). Adult porcine islets were obtained from two year old female Landrace-Yorkshire pigs (200 kg) purchased from the University of Alberta Farm, isolated using the same technique previously described for human islets, and cultured in the same medium as NPI (see above) (1, 21, 22). NOD-RAG−/− islets were isolated using established methods (23).
Islet Transplantation Studies
A single intraperitoneal (I.P.) injection of Streptozotocin (STZ, 200 mg/kg, Sigma) was administered to 8–10 week old male NOD-RAG−/− mice to induce diabetes, and animals were considered to be diabetic after two consecutive blood glucose measurements ≥325 mg/dL. Grafts containing a mass of 500 syngeneic NOD-RAG−/− islets, 2000 IEQ adult porcine islets, or 2000 NPI were transplanted under the left kidney capsule in confirmed diabetic mice. Animals were euthanized at 24 hr and seven days posttransplant, and the graft-bearing kidney was processed for immunohistochemical analysis.
In Vitro Hypoxia and Reoxygenation Injury Model
NPI that had been maintained in ordinary culture conditions for eight to nine days were shifted to <1% oxygen conditions using a hypoxia chamber system developed by R.T. Kilani et al. (24). Briefly, six-well plates containing 500 NPI/well were transferred to a modular-incubator chamber (Billups-Rothenberg, DeMar, CA) and placed inside a tissue culture incubator maintained at 37oC. A controlled purge for 15 min. (200 ml/sec.) replaced the surrounding air within the chamber with nitrogen containing 5% CO2. It has been determined previously that the final oxygen content within this system is 10 mmHg (≈1% O2) (24). Following 24 hr within the chamber, the plates were removed and either analyzed immediately or cultured for an additional 24 hr under normal conditions (20% O2, 5% CO2, 37oC) to simulate reoxygenation injury. NPI exposed to hypoxia or hypoxia with reoxygenation were compared to control NPI cultured for the same time period under normal conditions.
Cell Viability Assay
NPI were mildly dissociated (Enzyme-Free Dissociation Buffer, Invitrogen). Cell viability in metabolically active cells was measured by detecting the reduction of 3-(4,5-dimethylthiazolyl-2) 2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich Canada, Mississauga, Ontario) into a blue formazan product (25).
Apoptosis of islet cells following in vitro exposure to hypoxia or hypoxia with reoxygenation or alternately in vivo within transplanted grafts was quantified using TUNEL staining (Dead-end Apoptosis Detection System, Promega, Madison, WI). Nuclear counterstaining with DAPI (4′,6- diamino-2-phenylindol, Molecular Probes, Eugene, OR) was used to detect all cells present in the sample. For in vitro experiments, islets were mildly dissociated (Enzyme-Free Dissociation Buffer, Invitrogen), fixed with 4% paraformaldehyde, stained in suspension, applied directly to slides with coverslips, and analyzed immediately using fluorescent microscopy (UV excitation at 240–380 nm). Each sample was stained in triplicate, and at least 500 cells were counted per slide. Experimental samples were compared to non-treated control cultures to determine parallel rates of spontaneous apoptosis. For in vivo experiments, islet grafts were harvested, placed in formalin, processed and embedded in paraffin. Deparaffinized sections (10 μm) of embedded NPI or islet grafts were stained to identify β-cells using guinea pig anti-pig insulin antisera (1:1000, Dako, Carpinteria, CA) together with PE-conjugated goat anti-guinea pig IgG secondary antibody (1:200, Jackson Immunoresearch, West Grove, PA). Immediately following immunohistochemical staining for insulin, apoptotic nuclei were labeled with FITC-dUTP (Deadend Fluorometric TUNEL System, Promega, Madison, WI), and then slides counterstained with DAPI and analyzed by fluorescent microscopy. To quantify apoptosis in vivo, fields containing at least 500 cells were analyzed at 200× magnification. The number of TUNEL+ cells (green or yellow) within the insulin positive islet graft area of the section were counted and compared to the total number of DAPI+ nuclei within that same field to determine percent apoptosis. Sections were prepared from three transplant recipients in each cohort, and at least 4 fields were analyzed in each section.
Glucose-Stimulated Insulin Release Assay
For each experimental condition, duplicate aliquots containing 500 NPI were incubated in low-glucose medium (RPMI supplemented with 2 mM l-glutamine, 0.5% BSA, and 50.4 mg/dL glucose; all from Sigma-Aldrich Canada) for 10 min. The NPI were then washed twice with low glucose medium and resuspended in either low glucose medium or high glucose medium (RPMI supplemented with 2 mM l-glutamine, 0.5% BSA, and 360 mg/dL glucose) and incubated for 2h at 37oC, 5% CO2. Aliquots containing 250 μL supernatant from each sample were analyzed in triplicate for porcine insulin content using a radioimmunoassay (Linco Research, St. Charles, MO). To calculate the fold stimulation for each experimental condition, the mean insulin released from cells cultured in high glucose medium was divided by the mean insulin released from cells cultured in parallel in low glucose medium.
Western Blot Analysis
Lysates were prepared from 500 NPI or adult porcine islets in EDTA-free protease inhibitor cocktail (Roche) supplemented with 0.1% SDS. The bicinchoninic acid (BCA) assay was used to determine protein concentration (Pierce Chemical Co., Rockford, IL) in each sample, and Western blot analysis performed by running 20 μg total protein extract on a 10% SDS-PAGE gel, followed by transfer to nitrocellulose (Bio-Rad Laboratories, Hercules, CA). Nonspecific antibody-protein interactions were blocked using PBS containing 0.05% Tween-20 (PBS-T) and 5% skim milk powder (blocking buffer). X-linked inhibitor of apoptosis protein (XIAP) was detected using an anti-XIAP monoclonal antibody (Clone 48; BD Pharmingen, Mississauga, ON) at 1:250 dilution in blocking buffer. Lysates prepared from Hela cells, which naturally express high levels of XIAP, were used as a positive control. BCL-2 was detected using an anti-BCL-2 monoclonal antibody (Clone 124; Upstate, Lake Placid, NY) at 1:1000 dilution in blocking buffer. A positive control lysate prepared from Hela cells engineered to overexpress BCL-2 was generously provided by S. Wasilenko and M. Barry (University of Alberta, Edmonton, AB). Peroxidase-labeled goat anti-mouse IgG (1:2500 in blocking buffer, Amersham Pharmacia Biotech) was used to detect binding of each primary antibody to specific regions on the membrane. Blots were stained using Amido Black (Sigma-Aldrich Canada) to confirm that an equal amount of protein had been loaded in each lane and that the samples had not degraded prior to analysis.
SigmaPlot 2000 (SPSS, Inc. Chicago, IL, USA) was utilized for all statistical analysis in this study, and results are expressed as mean±SEM. Student's t-tests for paired and unpaired data were used to compare results in each experimental condition.
NPI Remain Viable and Do Not Undergo Apoptosis Following Hypoxia and Hypoxia-Reoxygenation Insult
The susceptibility of NPI to apoptosis during periods of hypoxia and following hypoxia-reoxygenation insult was examined in vitro using a hypoxic chamber apparatus that has previously been shown to induce high levels of apoptosis in a murine β-cell line (βTC-Tet) and in primary human islets (20, 26). Following exposure to hypoxia (<1% O2, 5% CO2) for 24 hr, >84% of NPI remained viable as measured by the MTT assay (Fig. 1A); this represents a modest and statistically insignificant decrease in signal as compared to untreated control NPI cultures (P=0.25). This minor loss in viability was reflected by a corresponding minimal increase in the number of apoptotic cells following hypoxia (10.57±1.60% TUNEL+ versus untreated control cultures with 7.62±4.06% TUNEL+ (P=0.54); Figure 1B). The apparent resistance of NPI to oxidative stress was further magnified when NPI were subjected to 24 hr of hypoxia followed by 24h of culture in normal oxygen conditions (20% O2, 5% CO2), to mimic hypoxia-reoxygenation injury. Levels of cell viability and rates of apoptosis in NPI cultures were statistically insignificant compared to those of control, untreated parallel cultures (Fig. 1A and 1B; p values of 0.29 and 0.65 respectively). Using the same experimental conditions, adult porcine islets were found to undergo high rates of spontaneous apoptosis in normal oxygen culture (50.03±10.33% TUNEL+) with virtually every cell dying following 24h of hypoxia (94.66±1.76% TUNEL+). Given the high rate of apoptosis following the hypoxic culture period, adult porcine islets were not exposed to a further 24 hr reoxygenation period. Control experiments for the chamber system were conducted using βTC-Tet cells in parallel, and these studies confirmed that the hypoxic chamber was functional and could induce cell death as reported previously (Fig. 1) (20, 26).
NPI Remain Moderately Glucose Responsive during Hypoxia and Recover Glucose Responsiveness/Insulin Production upon Reoxygenation
The earliest indicator of β-cell stress, which precedes the onset of apoptosis, is the loss of glucose responsiveness. To examine the extent of β-cell function remaining in NPI following hypoxia and hypoxia/reoxygenation, glucose-stimulated insulin release assays were performed immediately after each stressful condition, and values were compared to those found for untreated control cultures. Glucose- stimulated insulin release assays using adult porcine islets in parallel were not performed due to the high rate of spontaneous apoptosis (∼50%) and hypoxia-induced apoptosis (∼100%) observed in these islets in the hypoxia-reoxygenation model system. As shown in Figure 2A, there was a moderate decrease in the glucose stimulation index immediately following 24 hr of hypoxia (however this did not reach statistical significance compared to control cultures; P=0.19), whereas the stimulation index moved closer to control values following an additional 24 hr of reoxygenation. Figure 2B shows that although 24 hr of hypoxia reduced somewhat the capacity of NPI to secrete insulin in response to high glucose (Hypoxia 360 vs. Hyp. Control 360), a further 24 hr incubation under normal oxygen conditions allowed NPI to increase their capacity to secrete insulin in response to glucose (Reoxygenation 360 vs. Hypoxia 360). Thus, once reoxygenated, the insulin secretory capacity of hypoxic NPI appears to recover, although (at least in the first 24 hr) the rate of increase is somewhat lower than for control cultures which never received a hypoxic insult. This capacity to recover insulin secretion after a period of hypoxia followed by 24 hr of reoxygenation is very different from that observed for β-cell lines or human islets (20, 26).
NPI are Less Apoptotic during the Posttransplant Engraftment Period Compared to Adult Porcine Islets
To investigate the resistance of NPI to hypoxia-induced apoptosis in vivo, NPI were transplanted into chemically diabetic, immunodeficient NOD-RAG−/− mice and compared to adult porcine islet grafts. Islet grafts were harvested at 24 hr and seven days posttransplant in each cohort (n=3 at each time point) and processed for immunohistochemical analysis (Fig. 3; insulin staining in red, TUNEL staining in green, and costaining for insulin and TUNEL in yellow). As expected, adult porcine islet grafts were largely apoptotic 24 hr posttransplant, with 70.15±4.14% of the cells within the graft staining TUNEL+ (Fig. 3A, green and yellow staining). After seven days, when the remaining islets had engrafted, there was very little evidence of TUNEL+ apoptotic cells (1.20±0.11%). NPI grafts exhibited low levels of insulin staining at both 24 hr and seven days posttransplant, which is consistent with the finding that NPI generally require six to eight weeks to fully differentiate into a β-cell dense tissue (red insulin staining, Figure 3A) (8). However, there were significantly less TUNEL+ cells within NPI grafts 24 hr (8.74±2.82%) or seven days posttransplant (0.18±0.10%), compared to adult porcine islet grafts (Fig. 3B). In fact, the majority of cells that appeared to stain positive for TUNEL at 24 hr posttransplant were those that also stained positive for insulin (shown in yellow), suggesting that only the differentiated β-cells in NPI were sensitive to posttransplant apoptosis (Fig. 3A).
NPI Naturally Express High Levels the Potent Antiapoptotic Protein XIAP
To understand the mechanism by which NPI are resistant to apoptosis compared to adult porcine islets, lysates were prepared from NPI and adult porcine islets and analyzed by Western blot to determine expression levels of two different antiapoptotic proteins: BCL-2 and XIAP. BCL-2 is a potent inhibitor of mitochondrial-mediated apoptosis, which is the main intracellular apoptosis pathway implicated during oxidative stress-induced injury, while XIAP is an endogenous inhibitor of effector caspases that function downstream of both extracellular (receptor-mediated) and intracellular apoptotic pathways. Three different NPI preparations were compared to adult porcine islets, and a representative blot is shown in Figure 4. Neither NPI nor adult porcine islets expressed any detectable BCL-2 protein (upper panel). In contrast, NPI consistently exhibited high levels of XIAP expression, whereas XIAP could not be detected in adult porcine islets (lower panel). In similar Western blots we have been unable to detect endogenous XIAP expression in adult murine or adult human islets ([20, 26]; unpublished results).
The profound loss of mature islet mass in the early posttransplant engraftment period can be attributed to the onset of apoptosis due to the exquisite sensitivity of β-cells to hypoxia and reoxygenation injury. This study represents the first examination of the effects of oxidative stress on NPI, a widely available neonatal tissue that has the potential to be used as an alternative β-cell source in clinical islet transplantation. NPI were exposed to hypoxia and reoxygenation using an in vitro chamber model system which we have previously shown can induce significant loss of β-cell viability, loss of glucose responsiveness, and loss of overall mass due to apoptosis (20, 26). Surprisingly, the majority of NPI cells remained viable and did not become apoptotic during periods of hypoxia or even following reoxygenation (Fig. 1). In fact, in the face of hypoxia or reoxygenation NPI continued to secrete insulin in response to glucose (Fig. 2), the critical function that typically disappears first when β-cells experience stress. Several results in our study may provide insight into the maturation process of NPI in the context of islet transplantation. First, data shown in Figure 2B illustrates that once reoxygenated, the β-cell mass and/or insulin secretory capacity of NPI increases over time, irrespective of previous exposure to hypoxia. This suggests that for NPI in vivo, the hypoxic stress which occurs in the early posttransplant period will not necessarily have a significant impact on eventual graft maturation and function. The data obtained in our in vitro experiments (Figs. 1 and 2) are also consistent with our in vivo observation that there were relatively few apoptotic cells in NPI grafts at 24 hr posttransplant (Fig. 3). By one week posttransplant there were virtually no detectable apoptotic cells in NPI grafts, a result that has been observed by other groups (27).
In comparison to adult islet grafts, significantly fewer cells within NPI grafts were observed to undergo early posttransplant apoptosis. Thus NPI grafts will likely give rise to much less donor antigen that must be cleared by recipient antigen presenting cells, and we hypothesize that there should be less priming of the recipient's antigraft immune response. With islet grafts of any kind, the volume of tissue being transferred into the recipient is a tiny fraction of that transferred during a solid organ transplant (e.g. kidney); thus islets may hold greater promise for tolerance induction protocols, and particularly so if the islets undergo very little early posttransplant apoptosis. The inherent resistance of NPI to posttransplant hypoxia and reoxygenation injury makes them particularly attractive from this standpoint.
When we investigated the mechanism by which NPI exhibit robust survival in the face of significant stress, we found that NPI naturally express high levels of the potent anti-apoptotic gene XIAP, while islets from adult pigs do not (Fig. 4). This observation is consistent with the findings of Liggins et al., who observed that fetal human islets overexpress another IAP family member, Survivin, while adult human islets do not (28). IAP overexpression in noncancerous cells has only been observed in a few tissues, and appears to be temporally linked to specific developmental changes in these tissues (28–30). A temporal fall in the expression levels of potent endogenous antiapoptotic proteins in maturing islets may explain the observed neonatal wave of β-cell apoptosis which occurs in the first weeks of life in rodents (reviewed in ). The enhanced survival of NPI during periods of oxidative stress cannot be fully explained by XIAP overexpression alone because we have previously reported that although XIAP overexpression in murine β-cells and human islets potently inhibits hypoxia and hypoxia/reoxygenation-induced apoptosis, the functional response to glucose is transiently lost and recovers only slowly during the reoxygenation period (20, 26). The fact that NPI are nonapoptotic and remain glucose responsive suggests that the mechanism of NPI survival is multifaceted and complex.
In terms of mechanism, the behavior of NPI may be due to the fact that they consist of a mixture β- and other cells at various stages of differentiation. It is unlikely that the few highly differentiated β-cells present in NPI at the time of transplant are resistant to oxidative stress. Rather, we hypothesize that these cells rapidly succumb to hypoxia-induced apoptosis (similar to other fully differentiated β-cells), whereas the immature β-cells and pre-β-cells within the “NPI mixture” survive. Once reoxygenated, it is these latter cells that continue to differentiate to provide glucose-responsive insulin secretion. This idea is consistent with the result shown in Figure 3A, where within NPI grafts 24 h posttransplant, virtually all of the strongly insulin positive cells were also TUNEL positive, whereas scattered cells that stained light pink (containing only small quantities of insulin) were TUNEL negative. Also consistent with this hypothesis is the fact that for the insulin release experiments described in the left half of Figures 2A and 2B, hypoxia-treated cells were returned to an oxygenated environment during the incubation with high or low glucose. The possibility exists that the most desirable NPI preparations would be those which are devoid of any mature β-cells, but rich in pre-β-cells and/or immature β-cells to avoid apoptosis and immunological priming during transplantation.
In conclusion, our data indicate that NPI possess a natural resistance to hypoxia and reoyxgenation-induced apoptosis and loss of function in vitro, findings that were confirmed by our observation that NPI grafts had very little evidence of apoptosis during the early posttransplant period (24 hr) or thereafter (7 days). The robust survival of NPI compared to adult islets could be attributed in part to the overexpression of XIAP, a powerful inhibitor of effector caspases that functions late in apoptosis. However, there are probably additional factors that contribute to the hardiness of NPI, and further investigation into the mechanisms acting may well suggest new therapeutic strategies to enhance the survival of adult islets in the transplant setting. The resistance of NPI to apoptosis posttransplant implies that less donor antigen is released from NPI grafts compared to adult islet grafts, and as such NPI grafts may prove to be less immunogenic to the recipient. This concept is supported by the report that murine islet allografts engineered to overexpress XIAP were accepted long term in chemically diabetic recipients in the absence of immunosuppressive therapies (32). The recent finding that NPI allografts were spontaneously accepted in adult pigs in the absence of immunosuppression also supports this hypothesis (9). Thus, NPI represent an attractive alternative source of tissue to expand the availability of clinical islet transplantation, due to their nearly limitless availability, ease of preparation, and enhanced survival characteristics both in vitro and in vivo, as demonstrated by the present study.
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