Islet transplantation is considered an optional treatment of selected patients with type 1 diabetes (1, 2). Most islet transplant centers perform transplantation of cultured islets (3–6). The advantages of culturing islet preparations before transplantation over freshly isolated islets include (i) allowing the time for arranging the logistics of patient admission to the hospital, (ii) implementation of preconditioning therapy, and (iii) providing adequate time to assess the safety of the cell product by microbiological (i.e., mycoplasma, aerobes, and anaerobs cultures) and pyrogenic (endotoxin) tests. In addition, the development of a culturing system has allowed for the successful shipment of clinical human islet cell products to remote centers for transplantation (7, 8). The metabolic success measured after islet transplantation greatly depends on the mass of islets transplanted to the recipient (>13,000 islet equivalent [IEQ]/recipient kg in recent trials) and insulin independence is achieved generally after transplantation of more than one human preparation per recipient (1–8). Unfortunately, loss of islet mass during pretransplant culture (∼20%–30%) is one of the major issues requiring improvements to maximize the number of transplants and improve clinical outcomes (4). Implementation of improved culture conditions may be of assistance in preventing/reducing the loss of islet mass during pretransplant culture (9).
Pancreatic islet cells, especially β cells, undergo structural and functional alterations during pregnancy to cope with the increased insulin demand. The relationship between pregnancy and β-cell proliferation has been investigated to elucidate the mechanism of these changes (10–15). It has become clear that lactogens including prolactin (PRL), growth hormone (GH), and placental lactogens are involved in the phenomenon of β-cell proliferation. In addition, several recent studies demonstrated cytoprotective effects of PRL on insulin-producing cell lines and rodent islets treated by streptozotocin (STZ) in vitro (16–18) and in vivo (18). Treatment with GH and PRL protects the rat insulin-producing INS-1 cells from cytokine-induced apoptosis (19). Furthermore, in vivo studies in mice showed that PRL treatment significantly reduced the elevation of blood glucose levels in serum and the degree of insulitis in a model of STZ-induced diabetes (20). These results suggest that lactogen hormones may protect β cells against the noxious stimuli occurring during pancreas preservation, islet isolation, and culture for clinical transplantation.
The purpose of this study was to investigate the effects on human β cells of recombinant human PRL (rhPRL) supplementation to the culture media for clinical islet transplantation. Our study shows that rhPRL resulted in a significant improvement in β-cell survival during culture and also in protection of β cells against noxious stimuli in vitro. Moreover, surviving β cells demonstrated good functionality when transplanted into chemically induced diabetic immunodeficient mice. PRL supplementation to the culture media did not alter the production of proinflammatory mediators (cytokine/chemokine and tissue factor [TF]) during culture of islet preparations. These results suggest that PRL supplementation to culture media may represent a beneficial strategy in minimizing β-cell loss during pretransplant culture, which in turn could lead to an increase in successful islet transplantations.
MATERIALS AND METHODS
Human Islet Isolation and Culture
Pancreata were recovered from deceased multiorgan donors and then immediately placed in preoxygenated (30 min) two-layer perfluorocarbon/University of Wisconsin solution (21, 22) or with University of Wisconsin solution alone. The donor characteristics of 14 human pancreata used for this study are given in Table 1. Islets were isolated using the modified automated method (23) at the Human Cell Processing Facility of the Diabetes Research Institute's Cell Transplant Center at the University of Miami Miller School of Medicine. Islet yield and purity were determined by dithizone staining. Islet aliquots (3000 IEQ were cultured in Miami-defined culture medium (MM1; Mediatech-Cellgro, Herndon, VA) (7) with or without 500 μg/L of rhPRL (Sigma-Aldrich, St. Louis, MO) at 37°C for 2 days in 5% CO2 humidified incubator. After culture with or without rhPRL, islet samples were collected to count IEQ recovered and expressed as a percent of IEQ recovered over plated on day 0.
Assessment of Cellular Composition
As previously described (24), human islets were dissociated into single-cell suspensions using Accutase (Innovative Cell Technologies, San Diego, CA) for 10 min at 37°C. Dispersed cells were fixed on glass slides with 2.5% paraformaldehyde (Electron Microscopy Sciences, Washington, PA). To reduce nonspecific antibody binding, fixed cells were incubated overnight at 4°C with nondiluted Protein Block (BioGenex, San Ramon, CA). Subsequently, the cells were incubated for 2 hr at room temperature (RT) with primary antibodies: monoclonal mouse anti-C-peptide (1:100; Abcam Inc., Cambridge, MA); monoclonal mouse antiglucagon (1:500; Sigma-Aldrich); polyclonal rabbit antisomatostatin (1:500; Dako, Carpinteria, CA). After washing, samples were incubated at RT for 1 hr with AlexaFluor-488 goat anti-mouse IgG (1:200; Molecular Probes, Eugene, OR), Alexa Fluor 647 goat anti-rabbit IgG (1:200; Molecular Probes), and the nuclear-binding dye 4′,6-diamidino-2-phenylindole (1:300). Slides were analyzed using a immunocytofluorescence on dissociated cells by laser scanning cytometry (LSC/iCys; CompuCyte, Cambridge, MA) (24, 25).
Absolute β-, α-, and δ-cell mass after culture with or without rhPRL were calculated with following formulas (24–26):
Assessment of Fractional β-Cell Viability
After dissociation of islet aliquots using Accutase (see above), islet cell suspensions were incubated with 1 μM Newport Green PDX acetoxymethylether (NG; Molecular Probes) and 100 ng/mL of tetramethylrhodamine ethyl ester (Molecular Probes) for 30 min at 37°C in phosphate-buffered saline. After washing, cells were stained with 7-aminoactinomycin D (Molecular Probes) and then analyzed using a FACScan cytometer (Becton Dickinson, Mountain View, CA) with the CellQuest software, as described (24).
Assessment of β-Cell Proliferation
Aliquots of islets cultured with or without rhPRL (500 μg/L) were collected and frozen (−80°C) until assayed for Erk2 phosphorylation using fluorescence-based quantitative measurement on a Bio-Plex® system (BioRad, Hercules, CA), as described (27). Lysate protein concentration was determined by BioRad DC protein assay. Quantitative determination of phosphorylated proteins for Erk2 was carried out per manufacturer recommendations (BioRad). Data were calculated as ratio of phosphorylated Erk2-to-total Erk2. The value of percent control in the PRL group was shown.
BrdU and Ki-67 Staining
Islets were cultured with or without 500 μg/L of rhPRL at 37°C for 7 days in 5% CO2–humidified atmosphere. Culture media were exchanged every 2 days. To label newly synthesized DNA in dividing cells, 500 ng/mL of 5-bromo-2-deoxyuridine (BrdU; Roche, Laval, QC) was added to culture media 24 hr before the assessment. Islet cells were dispersed by Accutase and fixed on glass slides with 2.5% paraformaldehyde (see above) (24). Epitopes were retrieved by heat induction with Antigen Decloaker 10× (Biocare Medical, Concord, CA) in a rice cooker for 10 min at 120°C. After blocking nonspecific binding (Protein Block, 30 min, RT), cells were incubated for 2 hr at RT with mouse anti-BrdU (1:100; BD Biosciences, San Jose, CA) or mouse anti-Ki67 (clone MIB-1, 1:50; Dako), and anti-chicken insulin (1:500; Linco Research, St. Charles, MO). Then, the cells were labeled for 1 hr at RT with AlexaFluor-488 goat anti-mouse IgG (1:200) and AlexaFluor-647 goat anti-chicken IgG (1:200; all from Molecular Probes). The cell nuclei were stained twice for 10 min at RT with 4′,6-diamidino-2-phenylindole. The samples were analyzed using LSC/iCys (28, 29).
Delivery of Proapoptotic Stimuli to Islet Cells
Islet aliquots of 3000 IEQ were exposed for different periods of time to selected noxious stimuli to induce apoptosis 1 hr after preculture with or without 500 μg/L rhPRL. S-Nitroso-N-acetyl-dl-penicillamine (1.0 mM for 18 hr; Baxter Healthcare Corporation, Deerfield, IL) was used as a nitric oxide donor. Hydrogen peroxide (H2O2; 50 μM for 18 hr; Sigma) was used as a source of oxidative stress. Islet exposure for 24 hr to a cytokine cocktail (50 U/mL of interleukin [IL]-1β, 1000 U/mL of tumor necrosis factor [TNF]-α, and 1000 U/mL of interferon [IFN]-γ; all from R&D Systems, Minneapolis, MN) was used to mimic inflammation (30). After culture, islets were counted and processed for β-cell-specific viability and for cellular composition assessment (24).
Measurement of Inflammatory Mediators
Islet aliquots (500 IEQ in 0.5 ml) were cultured with or without 500 μg/L of rhPRL. After 24 hr, supernatant from islet preparations were collected to determine the concentrations of proinflammatory mediators; namely, IL-1 β, IL-6, IL-8, IFN-γ, monocyte chemoattractant (MCP)-1, macrophage inflammatory protein-1β, and TNF-α, using Multi-Plex cytokine kits following the manufacturer's protocol (Bio-Plex; Bio-Rad Laboratories) (26). Additionally, islets aliquots (500 IEQ) were homogenized, and TF was measured by ELISA (Imubind Tissue Factor; American Diagnostica, Greenwich, CT) (30). The amount of cytokines/chemokines and TF was normalized to total protein of the islet aliquot.
In Vivo Assessment of Islet Potency
Animal procedures approved by the Institutional Animal Care and Use Committee were performed at the Diabetes Research Institute's Preclinical Cell Processing and Translational Models Core. Athymic nu/nu (nude) mice (Harlan Laboratories, Indianapolis, IN) were housed at the Division of Veterinary Resources of the University of Miami School of Medicine in virus-antibody-free rooms using microisolated cages and with free access to autoclaved food and water. Animals were rendered diabetic via a single intravenous administration of 200 mg/kg of STZ (Sigma). Nonfasting blood glucose was assessed with a glucometer (OneTouch Ultra2, LifeScan, Milpitas, CA). Mice with sustained hyperglycemia (>300 mg/dL) were used as islet graft recipients. Human islet aliquots were cultured with or without rhPRL (500 μg/L) for 48 hr and then 1000 IEQ islets/mouse were transplanted under the left kidney capsule of nu/nu mice. Nonfasting blood glucose values were assessed after transplant, and reversal of diabetes was defined as stable nonfasting blood glucose less than 200 mg/dL. An intraperitoneal glucose tolerance test (2 g/kg dextrose in saline given after overnight fasting) was performed in selected animals to assess graft performance over 60 min (31). Nephrectomy of the graft-bearing kidney was performed in animals achieving normoglycemia after transplantation to confirm return to hyperglycemia and exclude residual function of the native pancreas (31).
Data are expressed as mean±SEM and analyzed using Excel for Windows, SigmaPlot, and GraphPad softwares for descriptive statistics and data plotting. Two samples were compared using Wilcoxon sign rank test or Student's t test; statistical significance was considered for p values less than 0.05.
Prolactin Improves Human β-Cell Survival During Culture
To investigate the effects of rhPRL on β-cell survival during culture, islet aliquots were cultured for 48 hr in conventional medium (MM1, control) with or without rhPRL. After culture, the recovery rate of islet in the PRL group was not significantly improved when compared with the control group (75.1%±8.8% vs. 70.2%±6.1%; p=n.s.). Fractional β-cell viability assays showed no statistically significant difference between the experimental groups (PRL vs. control, 59.1%±10.4% vs. 57.3%±11.2%; p=n.s.). The cellular composition was also evaluated using LSC/iCys to estimate overall β-cell mass. No significant differences were observed between islets in PRL and control groups when assessing α-cell (21.1%±3.1% vs. 22.9%±4.1%, respectively; p=n.s.) and δ-cell content (4.3%±1.1% vs. 4.3%±3.1%, respectively; p=n.s.). Conversely, the percentage of β cell in the PRL group was significantly higher than that in the control group (33.4%±3.5% vs. 23.9%±2.6%, respectively; P<0.05; Fig. 1A). The β-cell mass in the PRL group also resulted significantly higher when calculated based on the total protein of islet aliquots (control vs. PRL=124.5 vs. 171.0 μg, 137.3%±6.6% of control; P<0.05; Fig. 1B).
Effects of Prolactin on Human β-Cell Proliferation
We investigated the effects of rhPRL on the human islet cell proliferation during pretransplant culture. To this aim, Erk2 phosphorylation was assessed in islet aliquots cultured with or without rhPRL by the means of fluorescence-based quantitative measurement (Bio-Plex system). Erk2 phosphorylation in the PRL group was significantly higher than control (183.2%±39.7% of control, P<0.05; Fig. 2A). To further examine β-cell proliferation, human islets were cultured with MM1 in the presence or absence of rhPRL for 7 days. C-peptide+ BrdU+ or C-peptide+Ki67+ double-positive cells were quantitatively assessed using LSC/iCys. The percentage of C-peptide+BrdU+ or C-peptide+Ki67+ in the PRL group did not significantly increase when compared with the control group (BrdU: 2.22%±0.69% vs. 1.31%±0.37%, p=n.s; Ki67: 2.12%±0.52% vs. 1.24%±0.35%, p=n.s.; Fig. 2B). These results suggest that the higher number of surviving β cells in the PRL group during culture might not be caused by the β-cell proliferation with our protocol.
Prolactin Protects Human β-Cells From Noxious Stimuli
Prolactin is known for its cytoprotective properties. We investigated the cytoprotective effects of rhPRL on human β cells against noxious stimuli acting on different pathways of stress-induced islet cell death. Before injury, islets were precultured for 1 hr with or without rhPRL.
β-cell viability in islets treated with rhPRL was significantly improved in when compared with the control group after exposure to the cytokine cocktail (50.3%±1.3% vs. 45.2%±1.4%; P<0.05), nitric oxide donor (49.9%±1.0% vs. 45.4%±1.2%; P<0.05), and H2O2 (48.8%±1.7% vs. 40.1%±1.9%; P<0.05, respectively; Fig. 3). These results suggest that the cytoprotective effects of rhPRL might mainly contribute to higher β-cell survival during culture.
Effects of Prolactin on the Production of Inflammatory Mediators From Human Islets In Vitro
To examine the antiinflammatory effects of rhPRL supplementation to the culture media of human islet preparations, cytokine/chemokine production in the supernatant was evaluated after 48 hr of conventional culture. The production of IFN-γ, TNF-α, IL-1β, IL-6, IL-8, RANTES, MCP-1, and macrophage inflammatory protein-1β was comparable between the experimental groups (Fig. 4). In addition, there was no significant difference in TF levels in human islets (PRL vs. control: 20.1±4.8 pg/mL vs. 17.7±3.6 pg/mL, respectively; p=n.s.). The data suggest that rhPRL supplementation to culture media can improve β-cell survival without affecting proinflammatory mediators and TF production.
Prolactin Treatment of Human Islets Does Not Affect Potency In Vivo But Improves Long-Term Graft Function
To evaluate islet quality after 48 hr of culture with or without rhPRL, four independent human islet preparations were tested for in vivo islet potency test. After culture, islet aliquots of 1000 IEQ were prepared from both experimental groups and transplanted into chemically induced diabetic immunodeficient mice in (control group, n=10; PRL group, n=11). Seven of the 11 mice (63.6%) in the control group and five of the 10 mice (50.0%) in the PRL group reversed diabetes after transplantation (mean reversal time of 4.1±2.3 vs. 4.0±2.6 days, respectively, p=n.s.; Fig. 5A). Loss of graft function during the follow-up period was observed in two of the seven (28.5%) in the control group (on days 36 and 88), but in none (zero of the five, 0%) of the animals in the PRL group (p=n.s.; Fig. 5B). An intraperitoneal glucose tolerance test performed in the mice achieving normoglycemia showed comparable glucose clearance between the two experimental groups (Fig. 5C).
Numerous approaches have been proposed to improve culture conditions of islets (26, 30). Optimal cell culture conditions for islet transplantation should provide sufficient oxygen and nutrients to allow islet cells to recover from damages related to noxious insults induced by isolation, resulting in the reduction of islet cell loss. In this study, we investigated the possibility that rhPRL can be beneficial for pretransplant culture of islet cells. Our data show that rhPRL supplementation to the culture media significantly improved specifically β-cell survival. Overall, PRL seemed to improve β-cell survival without deteriorating islet quality and increasing immunogenicity (proinflammatory cytokine/chemokine and TF production). The unique cytoprotective properties of PRL in targeting specifically β cells may be of assistance in preserving viable β-cell mass during pretransplant culture and improving clinical outcome after islet transplantation.
A significant correlation has been recognized between β-cell mass and successful islet transplantation outcomes, which points to the fact that the estimation of transplanted β-cell mass may be important than that of the total islet quantity (namely, total IEQ) (24, 32). Many studies have reported that the cellular composition in human islet preparations significantly varies (24, 33, 34), possibly because of the different levels of vulnerability among islet cell subsets from stress and insults during the whole preislet transplantation process, including pancreas procurement, preservation, islet isolation, and culture. The characteristics and function of each cell subset composing islet preparations are indeed different. In particular, β cells may be particularly susceptible to oxidative stress resulting from the low antioxidant potential. Therefore, β-cell-specific protection from damages during whole preislet transplantation processes should be considered in the optimization of culture condition for islet transplantation.
Lactogen hormones such as PRL, GH, and placental lactogen are considered to have arisen from a common ancestral gene (12, 35). Lactogens increase during pregnancy leading to β-cell proliferation to adapt with increased fetal insulin demand (12). Only β cells, among the cells comprised in islets, are known to have PRL receptors (36, 37). Prolactin can stimulate β-cell proliferation; glucose-induced insulin release; insulin gene expression and biosynthesis in fetal, newborn, and adult rat islets (10, 14, 15); and INS-1 cells (13). It has been, in fact, recognized that the β cells undergo structural and functional modifications in pregnant rodents. Furthermore, β-cell-specific proliferation can be induced in rodent islets by means of extended culture in the presence of PRL (10). Moreover, the mitogenic effects of PRL have been observed in cultured human islets, although this requires long incubation periods (11) or culture on coated dishes (17). In our study, we could not confirm the significant increase of β-cell proliferation with BrdU and Ki-67 staining by PRL supplementation, probably because islets were incubated with the culture protocol conventionally used in the clinical settings of islet transplantation consisting of floating condition (no tissue-treated flasks) with media supplemented with human serum albumin (without fetal bovine serum). However, we observed significant improvement in β-cell-specific survival, which may be caused by the cytoprotective effects of PRL resulting in prevention of apoptosis, rather than the stimulation of the β-cell proliferation in our experimental conditions. Indeed, lactogens have been recognized to not only cause the β-cell proliferation but also have antiapoptotic effects on the β cells. A role for PRL in the regulation of cell death and survival has been observed in lymphoid cells (38). Fujinaka et al. (16) reported that lactogens, including PRL, directly protect rodent pancreatic β cells against the cytotoxic effects of STZ and dexamethasone. Many reagents that have cytoprotective effects on islet cells have been investigated and reported (39). Among them, nicotinamide ameliorates cellular damage caused by noxious stimuli such as hydrogen peroxide and a combination of proinflammatory cytokines in vitro (40). The use of nicotinamide during isolation and culture before transplantation has been shown to improve islet yields and islet quality by decreasing TF and MCP-1 production in human islet preparations (30). Those proinflammatory mediators have been negatively associated with clinical islet transplant outcomes (41, 42). Moreover, activations of c-jun N terminal kinase and nuclear factor-κB are triggers for the production of proinflammatory cytokines/chemokines that can impair islet-cell survival and function (43, 44). In addition, Emamaullee et al. (45) recently reported that prevention of apoptosis by pan-caspase inhibitor in vitro and in vivo significantly improved human islet graft functions and longevity in a mouse model. Therefore, targeted inhibitors of these proinflammatory pathways could be useful to protect islet cells from stress during preislet transplantation processing (46, 47). Furthermore, numerous peptide hormones relating to islets, such as glucagon-like peptide-1 (48), lactogens (12), hepatocyte growth factor (49), parathyroid hormone-related protein (50), and insulin-like growth factors, have been tested and promising results have been reported. However, many of these reagents and hormones improve viability of all the islet cell subsets in human islet preparations. Interestingly, in many cases, cytokine/chemokine production from islet preparations was also elevated, which may lead to β-cell damages through the direct toxic effect of cytokine/chemokine or via indirect effects by recruiting inflammatory cells to the transplant site. In our study, PRL did not increase proinflammatory cytokine/chemokine production from islet preparations. Pancreatic ductal cells are considered to be one of the main sources in cytokine/chemokine production and do not have PRL receptors. In fact, we found no effects of rhPRL on viability and content in this study (data not shown).
To improve clinical outcomes in islet transplantation, quick revascularization of islet grafts after transplantation is a key. When compared with pancreas transplantation, implanted islets have less blood perfusion because of no vessels in the early posttransplant period. Johansson et al. (18) demonstrated that PRL supplementation to culture media improves revascularization, blood perfusion, and oxygen tension of mouse and human islets implanted in immunodeficient mice. In addition, they have reported the beneficial effects of even systemic treatment of PRL using a rodent model. Although it is not realistic in a clinical setting when considering various influences of PRL throughout the organs, those results encourage us to consider the use of PRL during pretransplant culture in a clinical setting. The proper combination of cytoprotective reagents and hormones may allow for further improvements in clinical islet transplantation.
In conclusion, PRL supplementation to pretransplant culture media can specifically improve β-cell survival during culture through the prevention of apoptosis induced by insults from pretransplant islet processing without increasing proinflammatory mediators in human islet preparations. Moreover, the function of the surviving β cells remains intact as shown by in vivo potency into immunodeficient mice. Both in vitro and in vivo data in our study complement and extend recent reports of the cytoprotective properties of PRL for human islets (18).
β-Cell-specific cytoprotection by rhPRL supplementation to culture media may be of assistance in developing novel and efficient strategies to increase islet suitability for transplantation from a single donor pancreas with minimized risks or side effects.
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