Normoglycemic Nude Mice Research Articles

Extensive amyloid formation in transplanted microencapsulated mouse and human islets

Objective: Deposition of cell toxic islet amyloid is a frequent finding in type 2 diabetes and also in transplanted human islets, where it is a possible explanation for their long-term failure. One suggested reason for amyloid in transplanted islets is that their low vascular density results in a disturbed local clearance of islet amyloid polypeptide (IAPP). To test this hypothesis we analysed accumulation of amyloid in microencapsulated islets, which exemplify a non-vascularised islet graft. Methods: Isolated islets from human or transgenic mice expressing human IAPP were microencapsulated in alginate and cultured in vitro or transplanted under the kidney capsule of normoglycemic nude mice. The degree of amyloid was determined after Congo red staining and subcellular alterations were analysed with electron microscopy. Results: Insulin and IAPP secretion from transgenic mouse islets were markedly increased during stimulation with glucose after one week of culture, but encapsulated islets in general released less insulin. Amyloid was detected after both one and three weeks of culture in the transgenic mouse islets and the encapsulated islets were most affected. After transplantation, electron microscopy displayed both intra- and extracellular amyloid in microencapsulated as well as in non-encapsulated human and transgenic mouse islet grafts. However, amyloid was more frequent in the encapsulated grafts. Conclusion: Micro-encapsulation of pancreatic islets might serve as an important tool for studies of amyloid formation under enhanced circumstances.

Quantitative In Vivo Islet Potency Assay in Normoglycemic Nude Mice Correlates With Primary Graft Function After Clinical Transplantation

Reliable assays are critically needed to monitor graft potency in islet transplantation (IT). We tested a quantitative in vivo islet potency assay (QIVIPA) based on human C-peptide (hCP) measurements in normoglycemic nude mice after IT under the kidney capsule. QIVIPA was initially tested by transplanting incremental doses of human islets. hCP levels in mice were correlated with the number of transplanted islet equivalents (r(2) = 0.6, P<0.01). We subsequently evaluated QIVIPA in eight islet preparations transplanted in type 1 diabetic patients. Conversely to standard criteria including islet mass, viability, purity, adenosine triphosphate content, or glucose stimulated insulin secretion, hCP in mice receiving 1% of the final islet product was correlated to primary graft function (hCP increase) after IT (r(2)=0.85, P<0.01). QIVIPA appears as a reliable test to monitor islet graft potency, applicable to validate new methods to produce primary islets or other human insulin secreting cells.

Dexamethasone suppresses the expansion and transdifferentiation of transplanted porcine neonatal pancreas cell clusters (NPCCs) into β-cells in normal nude mice

This study was performed to investigate the effect of dexamethasone on the expansion and transdifferentiation of transplanted neonatal pancreas cell clusters (NPCCs) in vivo. Porcine NPCCs were generated from 1 to 3-day-old neonatal pigs. After transplantation (Tx) of 4000 islet equivalents (IEqs) of NPCCs beneath the renal subcapsular space of normoglycemic nude mice, dexamethasone (Dx, 1 mg/kg) or vehicles were injected daily. Intraperitoneal glucose tolerance testing (ip-GTT) was performed at 4 weeks (n = 4) and 10 weeks (n = 7) after Tx. After harvesting the grafts, total graft and beta-cell graft mass were determined by morphometric analysis. Although the mean value of AUCg was elevated in the Dx-treated group at 10 weeks after Tx, the glucose levels of all the animals by ip-GTT were within the normal range. At 10 weeks after Tx, the relative volume, absolute mass of beta-cells in the graft, and total graft mass were significantly lower in the Dx-treated group (relative volume of beta-cells: 22.0% versus 35.3%, P < 0.05; beta-cells mass: 1.0 +/- 1.2 mg versus 2.2 +/- 5.6 mg, P < 0.05, total graft mass: 4.4 +/- 5.4 mg versus 6.3 +/- 1.3 mg, P < 0.05, Dx-treated versus control), but there was no difference at 4 weeks. Morphologically prominent cystic structures were observed in the Dx group at 10 weeks. Our results suggest that dexamethasone suppresses the expansion and transdifferentiation of transplanted porcine NPCCs into beta-cells in normal nude mice.

Microcirculation of human pancreatic islets transplanted under the renal capsule of nude mice.

The aim was to measure the capillary blood pressure in transplanted human islets. Human islets were isolated at the Central Unit of the beta-cell Transplant in Brussels, Belgium. After transport to our laboratory, the islets were implanted under the renal capsule of normoglycemic nude mice. Two weeks later the capillary and venous blood pressures in the islet graft and adjacent renal parenchyma were measured with a micropuncture technique. Capillary blood pressure was approximately 5-8 mmHg in both graft and renal capillaries: twice as high as in native islets. Venous blood pressures were similar (4-5 mmHg) in the veins draining the graft and in the renal interlobular veins. All veins leading from the graft emptied into the renal parenchyma, that is, into interlobular veins. The capillary hypertension seen in transplanted human islets is probably necessary to secure adequate drainage through the renal veins. Whether this contributes to the poor results of long-term islet graft survival is unknown.

Increase in β-Cell Mass in Transplanted Porcine Neonatal Pancreatic Cell Clusters Is Due to Proliferation of β-Cells and Differentiation of Duct Cells*

A 20-fold increase in beta-cell mass has been found after transplantation of porcine neonatal pancreatic cell clusters (NPCCs). Here the mechanisms leading to this increased beta-cell mass were studied. NPCCs (4000 islet equivalents) generated after 8 days culture of digested neonatal pig pancreas were transplanted beneath the renal capsule of streptozotocin (STZ) diabetic and normoglycemic nude mice. Grafts were removed at 10 days, 6 weeks, and 20 weeks after transplantation for immunostaining and insulin content. Proliferation of beta-cells and duct cells was assessed morphometrically using double immunostaining for Ki-67 with insulin or cytokeratin 7 (CK7). Graft maturation was assessed with double immunostaining of CK7 and insulin. Apoptosis was determined using propidium iodide staining. beta-cell proliferation in NPCCs was higher after 8 days of culture compared with that found in neonatal pig pancreas. After transplantation, beta-cell proliferation remained high at 10 days, decreased somewhat at 6 weeks, and was much lower 20 weeks after transplantation. Diabetic recipients not cured at 6 weeks after transplantation had significantly higher beta-cell proliferation compared with those cured and to normoglycemic recipients. The size of individual beta-cells, as determined by cross-sectional area, increased as the grafts matured. Graft insulin content was 20-fold increased at 20 weeks after transplantation compared with 8 days cultured NPCCS: The proliferation index of duct cells was significantly higher in neonatal pig pancreas than in 8 days cultured NPCCs and in 10-day-old grafts. The incidence of apoptosis in duct cells appeared to be low. About 20% of duct cells 10 days post transplantation showed costaining for CK7 and insulin, a marker of protodifferentiation. In conclusion, the increase in beta-cell mass after transplantation of NPCCs is due to both proliferation of differentiated beta-cells and differentiation of duct cells into beta-cells.

Differentiation and maturation of porcine fetal islet cells in vitro and after transplantation.

Porcine fetal pancreas is a potential source of beta cells for transplantation. The immaturity of the cells is a problem. We have defined the optimal conditions for in vitro propagation of this tissue before transplantation. Porcine fetal pancreas tissue was obtained for tissue culture at various stages of development. Serum-containing and serum-free media and a variety of potential differentiation factors were tested. In vitro, the numbers of endocrine islet cells and their proliferation were quantified and functional maturity of the beta cells was assessed by perifusion. Growth and maturation of the cells was assessed 3 months after transplantation into nude mice. Highest beta cell mass was obtained from end-gestational, as compared with early fetal or neonatal, pancreas. Nicotinamide and sodium butyrate effectively increased the insulin content and the number of endocrine cells in culture. In combination, these factors led up to a 90-fold increase in the insulin content of islet-like cell clusters (ICC) as compared with untreated controls. However, a high level of cell death through apoptosis was observed in these maximally stimulated endocrine cells, and they did not survive as grafts when transplanted into nude mice. Instead, a serum-free culture medium containing 10 mM nicotinamide and 0.1 mM isobutylmethylxanthine was found to support both differentiation and proliferation of endocrine cells as loose ICCs. Insulin release from these ICCs was sensitive to glucose. When transplanted under the kidney capsule of normoglycemic nude mice, a high level of beta cell differentiation and function was evident only in the ICCs cultured in the serum-free medium, and in freshly isolated ICCs. When transplanted to hyperglycemic nude recipients, the cells cultured in serum-free medium for 3 weeks reversed hyperglycemia more consistently and rapidly than freshly isolated ICCs. Optimal maturation of porcine fetal pancreatic cells is obtained in serum-free medium supplemented with nicotinamide. Butyrate is a potent stimulus for beta cell differentiation but leads to increased apoptotic cell death.

Resistance of established porcine islet xenografts to humoral rejection by hyperimmune sera.

Although preformed natural antibodies cause hyperacute rejection of primarily vascularized xenografts, tissue grafts such as skin or islets are revascularized by in-growth of host capillaries and therefore might be resistant to circulating antibodies. We examined the effect of hyperimmune serum and primed T cells on the survival of long-term porcine islet xenografts in diabetic nude mice. Porcine islets were transplanted beneath the kidney capsule of streptozotocin-induced diabetic BALB/c athymic mice. Hyperimmune serum and sensitized splenocytes were prepared by repeated immunization of BALB/c mice with porcine lymph node cells. Splenic T cells were enriched by nylon wool column separation. Tissues were examined by immunohistology using murine- and porcine-specific monoclonal antibodies. Porcine islets survived in nude mice for > 100 days with high levels of circulating porcine C-peptide and maintenance of normoglycemia. Injection of the hyperimmune sera (IgG) into normoglycemic nude mice bearing porcine islets for > 70 days failed to induce rejection despite the continued presence of circulating anti-porcine cytotoxic antibody. Injection of sensitized T cells caused acute rejection of long-term (>140 days) porcine islets, whereas injection of naive T cells had no effect. Histologically, porcine islets removed from mice treated with hyperimmune serum showed no staining for IgG. Long-surviving porcine islet grafts showed strong staining for interleukin (IL)-10 and a lesser amount of IL-4 but no staining for IL-2 or interferon-gamma. Although fresh porcine islets were positive for swine leukocyte antigen class 1 antigen and intercellular adhesion molecule (ICAM)-1 but negative for mouse platelet endothelial cell adhesion molecule and ICAM-2, long-surviving porcine islets showed positive endothelial staining for mouse platelet endothelial cell adhesion molecule and ICAM-2. Established islet xenografts are resistant to hyperimmune serum as a result of a lack of target endothelial antigens, whereas they remain susceptible to rejection caused by primed T cells. Local production of Th2 cytokines may explain the inability of long-surviving islet xenografts to activate injected naive T cells.

RESISTANCE OF ISLET XENOGRAFTS TO HUMORAL REJECTION

122 Hyperacute rejection of primarily vascularized xenografts is caused by performed natural antibodies to the gal-α1,3-gal epitope expressed on the vascular endothelium. However, islet xenografts are revascularized by in-growth of host capillaries and therefore might be resistant to circulating antibodies. We examined the effect of hyperimmune serum and primed T cells on the survival of long-term islet xenografts in nude mice. Materials and Methods: Islets (104 IE) isolated from 6 month old Yorkshire pigs were transplanted beneath the kidney capsule of streptozocin-induced Balb/c nu/nu (nude) mice. Hyperimmune serum and sensitized splenocytes were prepared by repeated immunization of Balb/c mice with porcine lymph node cells. Splenic T cells were enriched by a nylon-wool column separation. Tissues were examined by immunohistology using specific monoclonal antibodies. Results: Porcine islets (PI) survived in nude mice for >100 days with high levels of circulating porcine C-peptide. When hyperimmune serum (IgG) which was cytotoxic to porcine lymphocytes at a 1/2048 dilution was injected into normoglycemic nude mice bearing PI for >70 days (n=6), no rejection was induced despite continued presence of circulating anti-porcine cytotoxic antibody up to 14 days after injection. In contrast, 5×106 primed T cells injected into mice bearing long-term PI (>140 days) caused acute rejection. Injection of 5×106 naive Balb/c T cells had no effect on the survival of longstanding PI. When nude mice were treated early (12 days) after islet transplantation, naive T cells, but not hyperimmune serum, caused acute rejection. Histologically, PI removed from mice treated with hyperimmune serum showed no staining for IgG whereas incubation of freshly isolated PI with hyperimmune serum resulted in heavy deposition of IgG within the islets. PI removed after rejection caused by injected T cells showed heavy cellular infiltration. Long-surviving xenografts showed strong staining for IL-10 and a lesser amount of IL-4, but no staining for IL-2 nor γIFN. While fresh PI were positive for SLA class I antigen and ICAM-1 but negative for mouse PECAM and ICAM-1, long surviving PI showed positive endothelial staining for mouse PECAM, ICAM-1 and ICAM-2. Conclusion: Islet xenografts are resistant to hyperimmune serum and probably to natural antibodies due to lack of target endothelium antigens in the grafts as a result of in-growth of host vessels while they remain susceptible to rejection caused by primed T cells. Local production of Th2 cytokines may explain the inability of long-surviving islet xenografts to activate transferred naive T cells.

Human pancreatic beta-cell deoxyribonucleic acid-synthesis in islet grafts decreases with increasing organ donor age but increases in response to glucose stimulation in vitro.

Human pancreatic beta-cell proliferation may be crucial for the success of islet transplantation. The aim of this study was to test the hypothesis that adult human beta-cells proliferate in vitro and in vivo and respond with increased rates of replication to factors known to promote rodent islet-cell proliferation, i.e. glucose, human recombinant GH, and FCS. For this purpose, human islets were prepared from a total of 19 adult heart-beating organ donors and cultured for 48 h with or without the additives described above. 3H-thymidine was added to the medium during the last 60 min of culture. After immunohistochemical staining for insulin and autoradiography, the labeling index (LI; i.e. % of labeled beta-cells over total number of beta-cells) was estimated by light microscopy. Islets also were transplanted under the kidney capsule of normal or alloxan-diabetic nude mice. After 2 weeks, 3H-thymidine was injected and the islet grafts prepared for determination of LI, as described above. Islets cultured at 5.6 mM glucose showed an increased beta-cell proliferation compared with islets cultured at 2.8 mM glucose (P < 0.05). However, culture at 11 mM glucose failed to further increase beta-cell proliferation. Addition of GH (1 microg/ml) to the medium, in the presence of 1% FCS and 5.6 mM glucose, did not influence the rate of beta-cell proliferation. In islets transplanted to hyperglycemic nude mice, beta-cell proliferation was similar to that observed in islets grafted into normoglycemic nude mice. Proliferation, however, decreased with increasing organ donor age. This study shows that pancreatic beta-cells from adult man are able to proliferate both in vitro and in vivo. Moreover, beta-cells from adult human donors respond with increased proliferation to glucose in vitro and show a decreased proliferation in vivo with increasing donor age.

A defective stimulus-secretion coupling rather than glucotoxicity mediates the impaired insulin secretion in the mildly diabetic F1 hybrids of GK-Wistar rats.

Adult F1 hybrids of male GK and female Wistar control rats exhibit mild, spontaneous non-insulin-dependent diabetes mellitus characterized by impaired glucose-induced insulin secretion. Using isolated pancreatic islets of hybrid rats, we first studied whether impaired glucose-induced insulin response is present not only in adult but also in neonatal rats. Furthermore, we investigated whether the impaired glucose-induced insulin response can be restored by long-term normalization of glycemia. Both 1-week- and 2- to 3-month-old hybrid rats had similar body weights but increased fed blood glucose levels (P < 0.05) compared with age-matched control rats. At 5.5 mmol/l glucose, insulin release was two- to threefold lower in isolated islets of hybrid than in control rats of both age groups (P < 0.05). At 16.7 mmol/l glucose, insulin secretion from hybrid islets was approximately 25% of that from control islets of both 1-week- and 2- to 3-month-old rats. For the second objective, batches of 250 islets from hybrid or control rats were transplanted under the kidney capsule of athymic, normoglycemic nude mice and maintained there for 4 weeks. Perfusion of kidneys demonstrated that glucose-induced (16.7 mmol/l) insulin secretion was impaired markedly in hybrid grafts compared with that in control grafts (0.66 +/- 0.23 vs. 1.8 +/- 0.38 pmol/20 min; P < 0.01), whereas stimulation by 20 mmol/l arginine resulted in similar insulin responses in both groups. The volumes of the grafted islets were similar in kidneys bearing either control or hybrid islets.(ABSTRACT TRUNCATED AT 250 WORDS)

Transplantation of fetal porcine pancreas to diabetic or normoglycemic nude mice. Evidence of a rapid engraftment process demonstrated by blood flow and heat shock protein 70 measurements.

The aims of the present investigation were to study the blood flow rate in transplanted fetal porcine islet-like cell clusters (ICC) with laser-Doppler flowmetry and to evaluate the influence of hyperglycemia on this process. In order to investigate the degree of stress in transplanted beta cells during engraftment, the amount of heat shock proteins (HSP) or stress proteins was measured. ICC were prepared from the fetal porcine pancreas by employing a tissue culture technique. A total volume of 6 microliters (approximately 750) ICC was transplanted under the left kidney capsule of either normo- or hyperglycemic C57BL/6J nude mice. The blood flow to the xenogeneic islet graft and the adjacent kidney parenchyma were measured with laser Doppler flowmetry (LDF). Within 2 hr after transplantation (day 0) or 3, 6, 9, 12 (both normo- and hyperglycemic recipients), 15, 40, or 85 (only normoglycemic recipients) days after implantation of ICC into the nude mice, the hsp70 contents of the grafts were analyzed by means of an immunoassay. Revascularization of the subcapsular ICC graft was initiated rapidly, and 3 days posttransplantation the nutritional blood supply constituted 70% of the adjacent kidney blood flow. This figure increased with time after implantation until the ICC graft had equal or even higher blood perfusion than the adjacent kidney parenchyma. Hyperglycemia in recipients did not affect the revascularization process. Hsp analysis from ICC in culture demonstrated a surprisingly high content of hsp70. However, 3-9 days after transplantation the hsp70 content was markedly reduced in both the normo- and hyperglycemic group and then remained low in both groups. Thus, the present study showed that ICC transplanted into normo- or hyperglycemic nude mice were rapidly revascularized, and that the production of hsp70 was lowered already 3 days after transplantation when compared with cultured ICCs.

Persistent impairment of the insulin response to glucose both in vivo and in vitro after streptozotocin exposure: Studies with grafted pancreatic islets and islets maintained in culture

The functional responses of the pancreatic B-cells after cytotoxic damage are still largely unknown. Using in vitro models to clarify this issue, we have recently observed a preferential reduction of glucose-stimulated insulin production and release in mouse pancreatic islets maintained in culture after in vitro exposure to streptozotocin. In order to evaluate the relevance of these findings in vivo, two sets of experiments were performed. First, mouse pancreatic islets were exposed in vitro to 2.2 mmol/l streptozotocin or vehicle alone, cultured for 6 days, and finally grafted under the kidney capsule of normoglycemic nude mice. Two weeks after transplantation there was no difference in the total DNA and insulin content between the two groups of islet grafts, but the insulin concentration, as expressed per microgram DNA, was decreased by 40% in the streptozotocin-treated islets. The insulin release of the grafts, during perfusion of the graft-bearing kidney in situ with 16.7 mmol/l glucose was diminished in the streptozotocin group, whilst perfusion with 16.7 mmol/l glucose plus 5 mmol/l theophylline was able partially to counteract the reduction in insulin release. In the second set of experiments, NMRI mice were injected iv with 160 mg/kg streptozotocin or vehicle alone, and their islets isolated 15 min after the injections. After 6 days in culture, there was no decrease in DNA, glucagon and somatostatin contents, but the insulin content was decreased by 40% in the streptozotocin exposed islets. These islets also showed a 60% decrease in the insulin response to glucose, which was partly counteracted by incubation with 16.7 mmol/l glucose plus 5 mmol/l theophylline.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of normoglycemia on the functional maturation of the human fetal beta cell.

Previous studies from this laboratory have shown that human fetal pancreas of gestational age 14-20 weeks was capable not only of growing but also of maturing in its insulinogenic response to an acute static challenge with glucose, once it was removed from its usual physiological environment and passaged for up to 37 weeks in the diabetic nude mouse (absolute age of 58 weeks, where absolute age = gestational age + duration of passage in the mouse). In order to analyze the dynamics of insulin secretion during this process of maturation, these experiments were repeated, the tissue being perifused after removal from the mouse. Normoglycemic nude mice rather than diabetic ones were used because of their hardier nature. Human fetal pancreata of gestational age 14-20 weeks were passaged in these normoglycemic animals, either subcutaneously or beneath the renal capsule, for 11 to 70 weeks (absolute age up to 86 weeks). The tissue was removed and, after a mean of 1 day in organ culture, perifused for 50 min with 20 mmol/L of glucose. While overall there was a slight but significant increase in insulin secretion over basal levels, there was no time-dependent maturation of the response to glucose, and no adult-type response at any stage, as occurs both physiologically and in tissue passaged in the diabetic nude mouse by this period.(ABSTRACT TRUNCATED AT 250 WORDS)