A model for human islet transplantation to immunodeficient streptozotocin-induced diabetic mice

Streptozotocin (STZ) is a cytotoxic glucose analogue that causes beta cell death and is widely used to induce experimental diabetes in rodents. The sensitivity of beta cells to STZ is species-specific and human beta cells are resistant to STZ. In experimental islet transplantation to rodents, STZ-diabetes must be induced before transplantation to avoid destruction of grafted islets by STZ. In human islet transplantation, injection of STZ before transplantation is inconvenient and costly, since human islet availability depends on organ donation and frail STZ-diabetic mice must be kept for unpredictable lapses of time until a human islet preparation is available. Based on the high resistance of human beta cells to STZ, we have tested a new model for STZ-diabetes induction in which STZ is injected after human islet transplantation. Human and mouse islets were transplanted under the kidney capsule of athymic nude mice, and 10-14 days after transplantation mice were intraperitoneally injected with five consecutive daily doses of STZ or vehicle. Beta-cell death increased and beta-cell mass was reduced in mouse islet grafts after STZ injection. In contrast, in human islet grafts beta cell death and mass did not change after STZ injection. Mice transplanted with rodent islets developed hyperglycemia after STZ-injection. Mice transplanted with human islets remained normoglycemic and developed hyperglycemia when the graft was harvested. STZ had no detectable toxic effects on beta cell death, mass and function of human transplanted islets. We provide a new, more convenient and cost-saving model for human islet transplantation to STZ-diabetic recipients in which STZ is injected after islet transplantation

Human serum versus human serum albumin supplementation in human islet pretransplantation culture: in vitro and in vivo assessment

There is conflicting evidence favoring both the use of human serum (HS) and of human serum albumin (HSA) in human islet culture. We evaluated the effects of HS versus HSA supplementation on 1) in vitro β-cell viability and function and 2) in vivo islet graft revascularization, islet viability, β-cell death, and metabolic outcome after transplantation. Islets isolated from 14 cadaveric organ donors were cultured for 3 days in CMRL 1066 medium supplemented with HS or HSA. After 3 days in culture, β-cell apoptosis was lower in HS group (1.41 ± 0.27 vs. 2.38 ± 0.39%, p = 0.029), and the recovery of islets was 77 ± 11% and 54 ± 1% in HS- and HSA-cultured groups, respectively. Glucose-stimulated insulin secretion (GSIS) was higher in HS group (29.4, range 10.4-99.9, vs. 22.3, range 8.7-70.6, p = 0.031). In vivo viability and revascularization was determined in HS- and HSA-cultured islets transplanted into the anterior chamber of the eye of Balb/c mice (n = 14), and β-cell apoptosis in paraffin-embedded mouse eyes. Islet viability and β-cell apoptosis were similar in both groups. Revascularization was observed in one graft (HS group) on day 10 after transplantation. Islet function was determined in streptozotocin (STZ)-diabetic nude mice (n = 33) transplanted with 2,000 IEQs cultured with HS or HSA that showed similar blood glucose levels and percentage of normoglycemic animals over time. In conclusion, human islets cultured in medium supplemented with HS showed higher survival in vitro, as well as islet viability and function. The higher in vitro survival increased the number of islets available for transplantation. However, the beneficial effect on viability and function did not translate into an improved metabolic evolution when a similar number of HSA- and HS-cultured islets was transplanted

Efectes de la sobreexpressió d’IGF2 en la protecció i la regeneració de les cèl·lules beta pancreàtiques

[cat]Un aspecte central, tant en el desenvolupament de la diabetis tipus 1 com en el de la tipus 2, és la reducció del nombre de cèl·lules beta productores d’insulina, i en ambdues condicions la pèrdua de massa beta s’ha atribuït en gran mesura a l’augment de la mort de les cèl·lules beta. A més, diversos treballs han posat de relleu la contribució fonamental de la replicació de les cèl·lules beta pel manteniment fisiològic i la regeneració de la massa de cèl·lules beta en models amb massa beta reduïda, la qual cosa pot suggerir que l’alteració de la replicació de les cèl·lules beta també pot contribuir de manera important a la reducció de la massa beta en la diabetis. El trasplantament d’illots és una teràpia prometedora per la curació de la diabetis mellitus, amb la que s’ha aconseguit un control acurat de la glucèmia de l’individu. Ara bé, el trasplantament d’illots presenta algunes limitacions importants, una de les principals és la baixa disponibilitat d’òrgans i la seva elevada demanda, agreujada per l’alt nombre d’illots requerits per restablir la normoglucèmia, probablement a causa de la important pèrdua de massa beta que té lloc els primers dies post-trasplantament. Aquesta pèrdua es deu als alts nivells d’apoptosi i de necrosi de les cèl·lules beta i a la seva limitada proliferació incapaç de compensar la mort. La interleucina 1 (IL-1) té un paper clau en la destrucció de les cèl·lules beta pancreàtiques que es dóna en la diabetis mellitus tipus 1 i tipus 2. A més, la citocina juga un paper important en l’increment de la mort beta que té lloc durant els primers dies després del trasplantament d’illots pancreàtics. El factor de creixement similar a la insulina 2 (IGF2) és un potent factor de creixement que té un rol important promovent la diferenciació i la proliferació cel·lular i actuant com a factor de supervivència en limitar l’apoptosi en diferents tipus cel·lulars. Per tant, IGF2 pot tenir un doble rol beneficiós en la massa de cèl·lules beta, actuant com a mitogen i com a factor de supervivència per les cèl·lules beta. La hipòtesi general del projecte és que el pronòstic del trasplantament d’illots pot millorar amb la utilització d’estratègies orientades a incrementar la supervivència i la replicació de les cèl·lules beta. L’objectiu general és determinar si la sobreexpressió d’IGF2 en els illots pancreàtics millora la supervivència i la regeneració de les cèl·lules beta pancreàtiques. L’objectiu de l’estudi in vitro fou caracteritzar l’efecte de la IL-1β sobre la replicació i supervivència de les cèl·lules beta i la potencial modulació per IGF2. L’objectiu de l’estudi in vivo fou determinar l’efecte de la sobreexpressió d’IGF2 sobre la massa beta trasplantada. En l'estudi in vitro, illots de rata control no infectats i illots infectats amb l’adenovirus que codifica per l’IGF2 (Ad-IGF2) es van cultivar durant 48h a 5,5 o 22,2 mM de glucosa en presència o absència de 1, 10, 30, i 50 U/ml d'IL-1β. La proliferació de les cèl·lules beta es va veure dràsticament reduïda quan els illots es van exposar a 10 U/ml d’IL-1β i es va suprimir gairebé completament en els illots exposats a 30 i 50 U/ml d’IL-1β. Concentracions més elevades de la citocina van ser necessàries per augmentar l’apoptosi de les cèl·lules beta de manera significativa. Malgrat la sobreexpressió d’IGF2 té un fort efecte mitogènic sobre les cèl·lules beta, IGF2 va preservar la replicació de les cèl·lules beta només en els illots exposats a 10 U/ml d’IL-1β, i no va tenir efecte en els illots exposats a altes concentracions de la citocina (30 i 50 U/ml). En contrast, la sobreexpressió d’IGF2 va presentar una clara protecció contra l’apoptosi induïda per IL-1β, pel que van ser necessàries concentracions superiors de la citocina per induir un augment significatiu de l’apoptosi respecte els illots control. Aquests resultats indiquen que la replicació de les cèl·lules beta és altament sensible als efectes deleteris de la IL-1β, com s’observa per la inhibició de la replicació per concentracions d’IL-1β relativament baixes, i la supressió quasi completa de la replicació de les cèl·lules beta per altes concentracions de la citocina. A més, els efectes inhibitoris de la IL-1β sobre la replicació de les cèl·lules beta no es van modificar per la glucosa, i van ser només modestament evitats per la sobreexpressió d’IGF2, en contrast amb la protecció proporcionada per la glucosa i per la sobreexpressió d’IGF2 contra l'apoptosi induïda per la IL-1β. En l'estudi in vivo, illots infectats amb l’adenovirus que codifica per l’IGF2 (grup Ad-IGF2), la luciferasa (grup control Ad-Luc), o amb illots no infectats (grup control) van ser trasplantats a rates Lewis diabètiques per estreptozotocina. Es van trasplantar 800 illots, que es una massa beta subcrítica, o 500 illots, que és una massa clarament insuficient per restaurar la normoglicèmia. Les rates trasplantades amb 800 illots que sobreexpressaven IGF2 van mostrar una millor evolució metabòlica respecte els grups control. Com era d'esperar, les rates trasplantades amb 500 illots que sobreexpressaven IGF2 o amb illots control van presentar una hiperglucèmia similar al llarg de tot l'estudi, assegurant condicions metabòliques comparables entre ambdós grups. La replicació de les cèl·lules beta va ser superior en el grup Ad-IGF2 vs el grup control els dies 3, 10, i 28 després del trasplantament. La massa beta es va reduir de manera similar el dia 3 després del trasplantament en el grup Ad-IGF2 i el grup control, es va incrementar el dia 10 i a dia 28 va ser superior en els empelts que sobreexpressaven IGF2. L'apoptosi es va incrementar de manera similar en ambdós grups, Ad-IGF2 i control, després del trasplantament. No es van trobar diferències en la secreció d'insulina entre els illots Ad-IGF2 i els illots control no infectats. En resum, la sobreexpressió de IGF2 en illots trasplantats va augmentar la replicació de cèl·lules beta, aquest increment de la replicació en els empelts d'illots que sobreexpressen IGF2 es va traduir en la regeneració de la massa beta trasplantada, fet que es reflectí en la millora del pronòstic del trasplantament. En conjunt, aquests resultats suggereixen que estratègies dirigides a augmentar la proliferació beta poden ser útils per induir la regeneració de la massa beta pel tractament de la diabetis i en el trasplantament d’illots.[eng]β-Cell mass reduction has a central role in the development of type 1 and type 2 diabetes, and in both conditions the loss of β-cells has been largely attributed to increased β-cell death. Furthermore several reports have highlighted the fundamental contribution of β-cell replication to the physiological maintenance of β-cell mass, and to β-cell mass regeneration in models with reduced β-cell mass. This may suggest that impaired β-cell replication could contribute to the reduction of β-cell mass in diabetes. Islet transplantation restores normoglycemia in type 1 diabetic patients. However, islet transplantation presents some important limitations. A basic restriction is the low organ availability and high requirement, which is exacerbated by the high islet mass that must be transplanted to achieve normoglycemia, probably due to the massive destruction of islets taking place in the initial days after transplantation due to increased beta-cell apoptosis, necrosi and impaired beta-cell replication. IL-1β is an important contributor to β-cell damage in type 1 diabetes, and recently it has also been related to the development of type 2 diabetes. IL-1beta also could contribute to the dramatic beta cell loss that takes place after islet transplantation IGF2 is a growth promoting peptide which is able to stimulate cell differentiation, proliferation and survival. Thus, IGF2 may play a dual beneficial role on β-cell mass, acting both as a mitogenic and as a survival factor for β-cells. We investigated in vitro and in vivo effects of the IGF2 overexpression on β-cell survival and regeneration. The aim of the in vitro study was to characterise the effect of IL-1β on β-cell replication, and the potential modulation by IGF2. Since the induction of β-cell proliferation by IGFs is dependent on ambient glucose concentration, low and high glucose concentrations were used to better define the effects of IL-1β and IGF2 on β-cell replication. The aim of the in vivo study was to determine the effect of IGF2 overexpression on β-cell mass in transplanted islets. In the in vitro study, control-uninfected and adenovirus encoding for IGF2 (Ad-IGF2)-infected rat islets were cultured at 5.5 or 22.2  mmol/l glucose with or without 1, 10, 30, and 50 U/ml of IL-1β. β-Cell replication was markedly reduced by 10 U/ml of IL-1β and was almost nullified with 30 or 50 U/ml of IL-1β. Higher concentrations of IL-1β were required to increase β-cell apoptosis. Although IGF2 overexpression had a strong mitogenic effect on β-cells, IGF2 could preserve β-cell proliferation only in islets cultured with 10 U/ml IL-1β, and had no effect with 30 and 50 U/ml of IL-1β. In contrast, IGF2 overexpression induced a clear protection against IL-1β-induced apoptosis, and higher concentrations of the cytokine were needed to increase β-cell apoptosis in Ad-IGF2-infected islets. These results indicate that β-cell replication is highly sensitive to the deleterious effects of the IL-1β as shown by the inhibition of replication by relatively low IL-1β concentrations, and the almost complete suppression of β-cell replication with high IL-1β concentrations. Likewise, the inhibitory effects of IL-β on β-cell replication were not modified by glucose, and were only modestly prevented by IGF2 overexpression, in contrast with the higher protection against IL-1β-induced apoptosis afforded by glucose and by IGF2 overexpression. In the in vivo study, islets infected with adenovirus encoding for IGF2 (Ad-IGF2 group), for luciferase (Ad-Luc control group), or with uninfected islets (control group) were syngeneically transplanted to streptozotocin-diabetic Lewis rats. Eight hundred islets, a minimal mass model to restore normoglycemia, or 500 islets, a clearly insufficient mass, were transplanted. Rats transplanted with 800 Ad-IGF2 islets showed a better metabolic evolution than control groups. As expected, rats transplanted with 500 Ad-IGF2 or control islets maintained similar hyperglycemia throughout the study, ensuring comparable metabolic conditions among both groups. β-Cell replication was higher in Ad-IGF2 group than in control group on days 3, 10, and 28 after transplantation. β-Cell mass was similarly reduced on day 3 after transplantation in Ad-IGF2 and control group, it increased on day 10, and on day 28 it was higher in Ad-IGF2 than in control group. Apoptosis was similarly increased in Ad-IGF2 and control islets after transplantation. No differences in insulin secretion were found between Ad-IGF2 and uninfected control islets. In summary, IGF2 overexpression in transplanted islets increased β-cell replication, induced the regeneration of the transplanted β-cell mass, and had a beneficial effect on the metabolic outcome reducing the β-cell mass needed to achieve normoglycemia. Taken together, these results suggest that strategies aimed to preserve or increase the engrafted β-cell mass may be useful for inducing the regeneration of beta mass for the treatment of diabetes and in islet transplantation

Increased beta cell replication, and beta cell mass regeneration in syngeneically transplanted rat islets overexpressing insulin-like growth factor-II

Insulin-like growth factor II (IGF2) is a growth-promoting peptide that increases β-cell proliferation and survival. The aim of the study was to determine the effect of IGF2 overexpression on β-cell mass in transplanted islets. Islets infected with adenovirus encoding for IGF2 (Ad-IGF2 group), for luciferase (Ad-Luc control group), or with uninfected islets (control group) were syngeneically transplanted to streptozotocin-diabetic Lewis rats. Eight hundred islets, a minimal mass model to restore normoglycemia, or 500 islets, a clearly insufficient mass, were transplanted. Rats transplanted with 800 Ad-IGF2 islets showed a better metabolic evolution than control groups. As expected, rats transplanted with 500 Ad-IGF2 or control islets maintained similar hyperglycemia throughout the study, ensuring comparable metabolic conditions among both groups. β-Cell replication was higher in Ad-IGF2 group than in control group on days 3 [1.45% (IQR: 0.26) vs. 0.58% (IQR: 0.18), p = 0.006], 10 [1.58% (IQR: 1.40) vs. 0.90% (IQR: 0.61), p = 0.035], and 28 [1.35% (IQR: 0.35) vs. 0.64% (IQR: 0.28), p = 0.004] after transplantation. β-Cell mass was similarly reduced on day 3 after transplantation in Ad-IGF2 and control group [0.36 mg (IQR: 0.26) vs. 0.38 mg (IQR: 0.19)], it increased on day 10, and on day 28 it was higher in Ad-IGF2 than in control group [0.63 mg (IQR: 0.38) vs. 0.42 mg (IQR: 0.31), p = 0.008]. Apoptosis was similarly increased in Ad-IGF2 and control islets after transplantation. No differences in insulin secretion were found between Ad-IGF2 and uninfected control islets. In summary, IGF2 overexpression in transplanted islets increased β-cell replication, induced the regeneration of the transplanted β-cell mass, and had a beneficial effect on the metabolic outcome reducing the β-cell mass needed to achieve normoglycemia

Increased beta cell replication, and beta cell mass regeneration in syngeneically transplanted rat islets overexpressing insulin-like growth factor-II

Insulin-like growth factor II (IGF2) is a growth-promoting peptide that increases β-cell proliferation and survival. The aim of the study was to determine the effect of IGF2 overexpression on β-cell mass in transplanted islets. Islets infected with adenovirus encoding for IGF2 (Ad-IGF2 group), for luciferase (Ad-Luc control group), or with uninfected islets (control group) were syngeneically transplanted to streptozotocin-diabetic Lewis rats. Eight hundred islets, a minimal mass model to restore normoglycemia, or 500 islets, a clearly insufficient mass, were transplanted. Rats transplanted with 800 Ad-IGF2 islets showed a better metabolic evolution than control groups. As expected, rats transplanted with 500 Ad-IGF2 or control islets maintained similar hyperglycemia throughout the study, ensuring comparable metabolic conditions among both groups. β-Cell replication was higher in Ad-IGF2 group than in control group on days 3 [1.45% (IQR: 0.26) vs. 0.58% (IQR: 0.18), p = 0.006], 10 [1.58% (IQR: 1.40) vs. 0.90% (IQR: 0.61), p = 0.035], and 28 [1.35% (IQR: 0.35) vs. 0.64% (IQR: 0.28), p = 0.004] after transplantation. β-Cell mass was similarly reduced on day 3 after transplantation in Ad-IGF2 and control group [0.36 mg (IQR: 0.26) vs. 0.38 mg (IQR: 0.19)], it increased on day 10, and on day 28 it was higher in Ad-IGF2 than in control group [0.63 mg (IQR: 0.38) vs. 0.42 mg (IQR: 0.31), p = 0.008]. Apoptosis was similarly increased in Ad-IGF2 and control islets after transplantation. No differences in insulin secretion were found between Ad-IGF2 and uninfected control islets. In summary, IGF2 overexpression in transplanted islets increased β-cell replication, induced the regeneration of the transplanted β-cell mass, and had a beneficial effect on the metabolic outcome reducing the β-cell mass needed to achieve normoglycemia

Increased beta cell replication, and beta cell mass regeneration in syngeneically transplanted rat islets overexpressing insulin-like growth factor-II

Insulin-like growth factor II (IGF2) is a growth-promoting peptide that increases β-cell proliferation and survival. The aim of the study was to determine the effect of IGF2 overexpression on β-cell mass in transplanted islets. Islets infected with adenovirus encoding for IGF2 (Ad-IGF2 group), for luciferase (Ad-Luc control group), or with uninfected islets (control group) were syngeneically transplanted to streptozotocin-diabetic Lewis rats. Eight hundred islets, a minimal mass model to restore normoglycemia, or 500 islets, a clearly insufficient mass, were transplanted. Rats transplanted with 800 Ad-IGF2 islets showed a better metabolic evolution than control groups. As expected, rats transplanted with 500 Ad-IGF2 or control islets maintained similar hyperglycemia throughout the study, ensuring comparable metabolic conditions among both groups. β-Cell replication was higher in Ad-IGF2 group than in control group on days 3 [1.45% (IQR: 0.26) vs. 0.58% (IQR: 0.18), p = 0.006], 10 [1.58% (IQR: 1.40) vs. 0.90% (IQR: 0.61), p = 0.035], and 28 [1.35% (IQR: 0.35) vs. 0.64% (IQR: 0.28), p = 0.004] after transplantation. β-Cell mass was similarly reduced on day 3 after transplantation in Ad-IGF2 and control group [0.36 mg (IQR: 0.26) vs. 0.38 mg (IQR: 0.19)], it increased on day 10, and on day 28 it was higher in Ad-IGF2 than in control group [0.63 mg (IQR: 0.38) vs. 0.42 mg (IQR: 0.31), p = 0.008]. Apoptosis was similarly increased in Ad-IGF2 and control islets after transplantation. No differences in insulin secretion were found between Ad-IGF2 and uninfected control islets. In summary, IGF2 overexpression in transplanted islets increased β-cell replication, induced the regeneration of the transplanted β-cell mass, and had a beneficial effect on the metabolic outcome reducing the β-cell mass needed to achieve normoglycemia

Pancreatic ductal cells may have a negative effect on human islet transplantation. PLoS One. 2019 Jul 19;14(7):e0220064.

Aim: To evaluate the effect of pancreatic ductal cells on experimental human islet transplantation. Materials and methods: Isolated islets were additionally purified by handpicking. Ductal cells were purified by magnetic cell sorting and then clustered into ductal pancreatospheres (DPS). Islets, DPS, and islets + DPS (100 islets + 75 DPS, or 100 islets + 200 DPS) were cultured and glucose-stimulated insulin secretion, β-cell apoptosis, and gene expression was determined. Islets and islets + DPS preparations (800 islets + 600 DPS) were transplanted to streptozotocin-treated immunodeficient mice and glycemia, graft morphometry, and gene expression were determined. Results: Insulin stimulation index was higher in islets than in islets co-cultured with DPS (5.59 ± 0.93 vs 4.02 ± 0.46; p0.05), and the ratio β-/endocrine non-β-cell mass was lower in islets + DPS grafts (islets: 2.05 ± 0.18, islets + DPS: 1.35 ± 0.15; p<0.01). IL1B and IL1RN expression was significantly higher in islets + DPS grafts. Conclusions: Islet preparations enriched with ductal cells have a lower insulin stimulation index in vitro and achieved a worse metabolic outcome after transplantation. Inflammation may mediate the deleterious effects of ductal cells on islet cells

Human serum versus human serum albumin supplementation in human islet pretransplantation culture: in vitro and in vivo assessment

There is conflicting evidence favoring both the use of human serum (HS) and of human serum albumin (HSA) in human islet culture. We evaluated the effects of HS versus HSA supplementation on 1) in vitro β-cell viability and function and 2) in vivo islet graft revascularization, islet viability, β-cell death, and metabolic outcome after transplantation. Islets isolated from 14 cadaveric organ donors were cultured for 3 days in CMRL 1066 medium supplemented with HS or HSA. After 3 days in culture, β-cell apoptosis was lower in HS group (1.41 ± 0.27 vs. 2.38 ± 0.39%, p = 0.029), and the recovery of islets was 77 ± 11% and 54 ± 1% in HS- and HSA-cultured groups, respectively. Glucose-stimulated insulin secretion (GSIS) was higher in HS group (29.4, range 10.4-99.9, vs. 22.3, range 8.7-70.6, p = 0.031). In vivo viability and revascularization was determined in HS- and HSA-cultured islets transplanted into the anterior chamber of the eye of Balb/c mice (n = 14), and β-cell apoptosis in paraffin-embedded mouse eyes. Islet viability and β-cell apoptosis were similar in both groups. Revascularization was observed in one graft (HS group) on day 10 after transplantation. Islet function was determined in streptozotocin (STZ)-diabetic nude mice (n = 33) transplanted with 2,000 IEQs cultured with HS or HSA that showed similar blood glucose levels and percentage of normoglycemic animals over time. In conclusion, human islets cultured in medium supplemented with HS showed higher survival in vitro, as well as islet viability and function. The higher in vitro survival increased the number of islets available for transplantation. However, the beneficial effect on viability and function did not translate into an improved metabolic evolution when a similar number of HSA- and HS-cultured islets was transplanted