Fetal Porcine Islet-like Cell Clusters Research Articles

Xenotransplant issues

To determine the role of the terminal α-galactosyl residue in the endothelial damage mediated by human xenoreactive natural antibodies (IgM and IgG), we treated porcine endothelial cells in culture with green coffee bean α-galactosidase. A practically complete removal of terminal α-Gal residues (as evaluated by flow cytometry with Bandeiraea simplicifolia isolectin B4) and concomitant exposure of N-acetyllactosamine were obtained without altering cell viability. A dramatic decrease in IgM and IgG binding (from a pool of human sera) was observed, confirming the key role of the α-galactosyl residues. The enzyme treatment did not induce any nonspecific immunoglobulin binding sites but led to the exposure of new epitopes for a minor fraction of IgM. The main residual IgM and IgG binding could be due to xenoantigens other than the α-galactosyl residues. When α-galactosidase-treated endothelial cells were used as targets in cytotoxicity experiments, they were less susceptible than untreated cells to complement-mediated cytotoxicity induced by fresh human serum. In contrast, they did not acquire resistance to human IgG-dependent cellular cytotoxicity, despite the decrease in IgG binding. Because it is known that antibody-dependent cytotoxicity mediated by CD 16 NK cells is dependent on IgG1 and IgG3, and not IgG2 or IgG4, which was confirmed by blocking experiments, we studied the binding of all four subclasses to intact and α-galactosidasetreated endothelial cells. Two major subclasses, IgG1 and IgG2, bound to untreated endothelial cells, whereas IgG3 binding was low and IgG4 binding was negligible. A decrease in IgG1, IgG2, and IgG3 binding was observed upon α-galactosidase treatment, indicating that antibodies belonging to these three subclasses recognize α-galactosyl residues. The decrease in IgG2 binding was more pronounced than the decrease in IgG1 binding. Collectively, these data indicate that IgG1 xenoreactive natural antibodies, including those which are not directed at the α-galactosyl residues, could play a major role in the early delayed vascular rejection of pig xenografts. Fetal porcine islet-like cell clusters (ICC) were transplanted under the renal capsule of normoglycemic normal or athymic (nu/nu) C57BL/6 mice. Control animals were implanted with allogeneic minced kidney tissue from C57BL/Ks mice. The animals were killed 6 or 14 days after transplantation and the grafts were processed for flow cytometric analyses or immunohistochemistry. Xenograft destruction was evident in normal mice on day 6 after transplantation. The majority of infiltrating cells were macrophage-like cells expressing the F4/80 antigen. Lymphocytes expressing the CD3 antigen. were in the minority and mainly located in the peripheral parts of the ICC xenograf. The frequency and distribution of CD4+ cells were found to resemble those of the CD3+ cells. A large number of infiltrating cells, including several macrophage-like cells, expressed the Thy 1.2 antigen. Flow cytometry of infiltrating cells in the ICC xenograft revealed that approximately half of the cells expressing the F4/80 antigen also expressed Thy 1.2 and/or CD4. No cells were found expressing both the F4/80 and CD8 antigens. Both the F4/80 single-positive and the F4/80, CD4 doublepositive cells were found to be large and more granular than the CD4 single-positive cells No co-expression of CD4 or Thy 1.2 with the F4/80 antigen was detected on cells infiltrating allogeneic tissue grafts. Moreover, a relative large number of cells (=15%) in the xenograft expressed the NK 1.1 antigen as determined by flow cytometry. The role of natural killer (NK) cells in islet xenograft rejection was further evaluated in mice depleted of NK cells, using intraperitoneal injections of the monoclonal antibody NK 1.1. The simultaneous inoculation and subsequent growth of the NK cell-sensitive β2- microglobulin-deficient mutant, C4.4–25, lumphoma cell line EL-4, served as an in vivo control of NK cell depletion. However, all NK cell-depleted mice rejected the ICC xenograft. In contrast, athymic mice permanently accepted the porcine ICC xenograft but readily rejected the NK cell-sensitive lymphoma cell line. taken together, ICC xenograft rejection in mice seems to be T Cell-dependent, as evidenced in the nude mice model, while the main effector cel appears to be a macrophage with a unique phenotype.

Reinnervation of transplanted fetal porcine endocrine pancreas. Evidence for initial growth and subsequent degeneration of nerve fibers in the islet grafts.

Syngeneic mouse islets were transplanted under the renal capsule of athymic nude (nu/nu) C57BL/6 mice. Likewise, neonatal rat islets or fetal porcine islet-like cell clusters (ICC) were transplanted into nude mice. The animals were killed at various times after implantation and the graft-bearing kidney was removed. The transplant was processed for microscopic examination with indirect immunofluorescence for neuropeptides and tyrosine hydroxylase, and with acetylcholine esterase staining to visualize nerve fibers in the graft. In all grafts, reinnervation with both afferent and efferent nerve fibers was encountered 20 weeks after transplantation. The pattern of reinnervation was quantitatively and qualitatively independent of the source of the implanted islets. These findings indicate that the pattern of reinnervation depends on the implantation organ and not on any inherent properties of the implanted endocrine cells. In addition, surviving vasoactive intestinal peptide-positive neurons within the fetal porcine ICC demonstrated an active ingrowth of nerve fibers into the ICC graft and the adjacent kidney parenchyma after transplantation. However, 12 or 16-24 months after transplantation, marked atrophy of all types of nerve fibers in the ICC grafts was observed. The reason for this late degeneration of nerve fibers is unknown, but it may be related to a failure to establish functional neural connections.

Xenograft rejection of porcine islet-like cell clusters in normal and natural killer cell-depleted mice.

Fetal porcine islet-like cell clusters (ICC) were transplanted under the renal capsule of normoglycemic normal or athymic (nu/nu) C57BL/6 mice. Control animals were implanted with allogeneic minced kidney tissue from C57BL/Ks mice. The animals were killed 6 or 14 days after transplantation and the grafts were processed for flow cytometric analyses or immunohistochemistry. Xenograft destruction was evident in normal mice on day 6 after transplantation. The majority of infiltrating cells were macrophage-like cells expressing the F4/80 antigen. Lymphocytes expressing the CD3 antigen were in minority and mainly located in the peripheral parts of the ICC xenograft. The frequency and distribution of CD4+ cells were found to resemble those of the CD3+ cells. A large number of infiltrating cells, including several macrophage-like cells, expressed the Thy 1.2 antigen. Flow cytometry of infiltrating cells in the ICC xenograft revealed that approximately half of the cells expressing the F4/80 antigen also expressed Thy 1.2 and/or CD4. No cells were found expressing both the F4/80 and CD8 antigens. Both the F4/80 single-positive and the F4/80, CD4 double-positive cells were found to be larger and more granular than the CD4 single-positive cells. No co-expression of CD4 or Thy 1.2 with the F4/80 antigen was detected on cells infiltrating allogeneic tissue grafts. Moreover, a relative large number of cells (approximately 15%) in the xenograft expressed the NK 1.1 antigen as determined by flow cytometry. The role of natural killer (NK) cells in islet xenograft rejection was further evaluated in mice depleted of NK cells, using intraperitoneal injections of the monoclonal antibody NK 1.1. The simultaneous inoculation and subsequent growth of the NK cell-sensitive beta 2-microglobulin-deficient mutant, C4.4-25-, lymphoma cell line EL-4 served as an in vivo control of NK cell depletion. However, all NK cell-depleted mice rejected the ICC xenograft. In contrast, athymic mice permanently accepted the porcine ICC xenograft but, readily rejected the NK cell-sensitive lymphoma cell line. Taken together, ICC xenograft rejection in mice seems to be T cell dependent, as evidenced in the nude mice model, while the main effector cell appears to be a macrophage with a unique phenotype.

Toxicity in vivo of different immunosuppressive drugs in fetal porcine pancreatic islets.

Clinical transplantation of fetal porcine islet-like cell clusters (ICC) to kidney transplanted diabetic patients has shown both morphological and biochemical evidence of prolonged xenograft survival, but no effect was seen on the insulin requirement of the transplanted patients. One possible explanation for this relative lack of success might have been the influence of the immunosuppressive drugs on the differentiation of ICC grafts. In this study, the effects of a number of immunosuppressive drugs on ICC differentiation were investigated. Normoglycemic C57BL/6 nu/nu mice were transplanted with 2x3 microliter ICC under the renal capsule. During a four-week period the animals were treated daily with azathioprine (2.0 mg/kg b.wt.), prednisolone (0.5 mg/kg b.wt.), cyclosporine (5.0 mg/kg b.wt.), cyclophosphamide (5.0 mg/kg b.wt.) 15-deoxyspergualin (5.0 mg/kg b.wt.), leflunomide (30 mg/kg b.wt.) or saline. In order to estimate rates of beta-cell DNA synthesis in the ICC grafts the mice were injected with 3H-thymidine one hour before killing and slides of the grafts were evaluated with regard to autoradiographical labeling. Other ICC grafts were evaluated by measurement of their insulin and DNA contents. Both the DNA content of ICC grafts and the beta cell labeling index in the cyclosporine animals were significantly decreased. Perfusion experiments with graft-bearing kidneys of cyclosporine-treated animals showed a significantly decreased insulin secretion in response to glucose plus theophylline. None of the other drugs influenced the differentiation of grafted ICC as evaluated in this study. Thus, it is obvious that cyclosporine inhibits both the growth and functional differentiation of transplanted ICC, which might be one reason for the relative lack of success in the clinical transplantation of porcine ICC to diabetic patients.

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.

Transplantation of porcine fetal pancreas to diabetic patients

Transplanation of fetal porcine islet-like cell clusters (ICC) reverses diabetes in experimental animals. We have now transplanted porcine ICC to ten insulin-dependent diabetic kidney-transplant patients. All patients received standard immunosuppression and, at ICC transplantation, antithymocyte globulin or 15-deoxyspergualin. ICC were injected intraportally or placed under the kidney capsule of the renal graft. Four patients excreted small amounts of porcine C-peptide in urine for 200-400 days. In one renal-graft biopsy specimen, morphologically intact epithelial cells stained positively for insulin and glucagon in the subcapsular space. We conclude that porcine pancreatic endocrine tissue can survive in the human body.

Polyamine requirements in nicotinamide-stimulated beta-cell differentiation in fetal porcine islet-like cell clusters.

The impact of nicotinamide on regulation of cell replication and hormone production by fetal porcine islet-like cell clusters (ICCs) containing a low fraction of beta-cells was investigated. For this purpose, ICCs were produced in tissue culture for 4 days in the presence or absence of nicotinamide (10 mM). ICCs formed in the presence of nicotinamide showed increased rates of (pro)insulin biosynthesis, contained more insulin, and displayed elevated contents of polyamines when compared with untreated ICCs. Immunocytochemical analyses of autoradiographed ICCs disclosed that nicotinamide expanded the relative size of the beta-cell population, and that the vitamin increased DNA synthesis of non-beta-cells only. The possibility that the effects of nicotinamide were conveyed by the increased polyamine content was explored by giving nicotinamide together with inhibitors of rate-limiting enzymes of polyamine biosynthesis, a maneuver that precluded the increases in polyamines of whole ICCs. Although this treatment prevented the increases in beta-cell population size, insulin production, and insulin content evoked by nicotinamide, the elevated cell replication nonetheless persisted. We conclude that the stimulatory effects of nicotinamide on insulin production and content by fetal porcine ICCs result from neoformation of beta-cells through differentiation. Since these effects were prevented by blocking the increased polyamine content, it is suggested that a stimulated synthesis of polyamines may be an important event in mediating the differentiating action of nicotinamide.

Polyamine requirements in nicotinamide-stimulated beta-cell differentiation in fetal porcine islet-like cell clusters

Polyamine requirements in nicotinamide-stimulated beta-cell differentiation in fetal porcine islet-like cell clusters

Treatment with an interleukin-1 receptor antagonist protein prolongs mouse islet allograft survival.

An interleukin-1 receptor antagonist protein was evaluated with regard to its efficacy in allogeneic and xenogeneic islet transplantation. Alloxan-induced diabetic C57BL/6 (H-2b) mice were transplanted under the kidney capsule with 500 C57BL/Ks (H-2d) mouse islets. Alzet osmotic pumps, which release their content over an 11- to 13-day period, were implanted subcutaneously for continuous infusion of interleukin-1 receptor antagonist protein (1.0, 5.0, 8.0 mg.kg-1 x day-1) or phosphate-buffered saline. Blood glucose determinations were performed every second or third day; at death, the islet-bearing kidneys were morphologically evaluated. Mice treated initially with the higher interleukin-1 receptor antagonist protein concentrations were followed for an additional period after cessation of the drug release to evaluate whether a transitory interleukin-1 receptor antagonist protein treatment would induce tolerance to the graft. All phosphate-buffered saline-treated mice were hyperglycemic 11 days after islet allotransplantation. Most of their grafts were heavily infiltrated with mononuclear cells. In the various interleukin-1 receptor antagonist protein-treated groups, 60-80% of the mice were normoglycemic after 11 days. Moreover, light microscopic examinations showed that most mice treated with interleukin-1 receptor antagonist protein had normal islet grafts or grafts infiltrated with only a few mononuclear cells. After interruption of interleukin-1 receptor antagonist protein infusion (8.0 mg.kg-1 x day-1), all animals developed hyperglycemia within 2-9 days. In xenogeneic experiments, 500-750 fetal porcine islet-like cell clusters were transplanted under the kidney capsule of normoglycemic C57BL/6 mice. These animals were treated either with interleukin-1 receptor antagonist protein (8.0 mg.kg-1 x day-1) or phosphate-buffered saline.(ABSTRACT TRUNCATED AT 250 WORDS)

INJECTION OF XENOGENEIC ENDOCRINE PANCREATIC TISSUE INTO THE PORTAL VEIN—EFFECTS ON COAGULATION, LIVER FUNCTION, AND HEPATIC HEMODYNAMICS

The safety of injecting discordant xenogeneic fetal endocrine pancreatic tissue into the portal vein was studied in a pig-to-dog system. It was found that minced fetal porcine pancreas and fetal porcine isletlike cell clusters prepared by collagenase digestion and culture could be injected with only minor or no hepatic hemodynamic disturbances. Coagulation studies revealed a small increase in plasma fibrinopeptide A, but this increase could be prevented by heparinization of the recipient. There was no consumption of fibrinogen or platelets. In contrast, injection of minced adult porcine pancreas caused pronounced hepatic hemodynamic changes and marked coagulation abnormalities, indicating consumption coagulopathy. The present finding that fetal porcine pancreas can be injected intraportally without deleterious effects in dogs provides a foundation for the eventual clinical use of such material as treatment for insulin-dependent diabetes mellitus.

Functional and morphological differentiation of fetal porcine islet-like cell clusters after transplantation into nude mice

By using a previously described culture technique for the midgestational fetal porcine pancreas, islet-like cell clusters with a Beta-cell frequency of approximately 5% have been produced in large numbers. These islet-like cell clusters were transplanted beneath the kidney capsule to either normoglycaemic or alloxan-treated nude mice. The grafts consistently failed to cure the alloxan-treated mice immediately after implantation, however, normoglycaemia was restored in a majority of the mice within 2 months after transplantation and in all animals after 4 and 6 months. Indeed, the insulin released from the transplanted fetal Beta cells was able to normalize the serum glucose concentration at porcine levels (4-5 mmol/l) rather than at the level maintained in mice (8-10 mmol/l). In the cured mice there was a normal secretory response to glucose in the grafts as evidenced by normal glucose profiles during intravenous glucose tolerance test and a biphasic insulin response to high glucose when perfusing the graft bearing kidney. On the other hand, in the normoglycaemic animals the second phase faded before the glucose stimulus had been withdrawn. Two months after transplantation the endocrine cells were arranged so that the endocrine non-Beta cells were randomly scattered among a majority of Beta cells. The cell replication of the Beta cells, measured by 3H-thymidine incorporation, was within the lower range of that seen in the native islets of adult mice. No major differences between the controls and the alloxan-treated animals were observed in this respect. Cultured islet-like cell clusters had high rates of glucose utilization, paralleled by low rates of glucose oxidation, compared with adult mouse islets.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Culture Conditions on Formation and Hormone Content of Fetal Porcine Isletlike Cell Clusters

To establish methods for stimulation of the growth and differentiation of fetal endocrine pancreatic cells, a technique for the in vitro production of fetal porcine isletlike cell clusters (ICCs) was used. By varying the composition of the culture medium with different glucose concentrations and the addition to the culture medium of insulin, growth hormone (GH), amino acids, or nicotinamide, we estimated the formation of ICCs and their hormone content. High glucose content (28.0 mM) stimulated the formation of abundant ICCs that contained decreased amounts of insulin. In contrast, culture at a low (5.6-mM) glucose concentration increased the ICC insulin content but decreased the number of ICCs formed. Addition of seven times the normal amount of amino acids hampered both the formation of ICCs and their insulin content. Neither insulin nor GH supplementation of the medium influenced the ICC insulin content, but GH stimulated an abundant outgrowth of ICCs containing relatively high insulin concentrations. However, ICCs formed under these circumstances contained less than 10% of the insulin content of adult islets, and further work has to be carried out to identify factors responsible for further differentiation of the fetal porcine pancreas.

Large-scale production of fetal porcine pancreatic isletlike cell clusters. An experimental tool for studies of islet cell differentiation and xenotransplantation.

A recently described method for the preparation of isletlike cell clusters (ICC) from human fetal pancreas has been applied to the fetal pig with the ultimate aim of large-scale production of ICC. Fetuses ranging in age from 51 to 77 days were used, and after a brief collagenase-incubation the pancreatic digest was plated into culture dishes containing medium RPMI 1640 supplemented with either 10% fetal calf serum (FCS) or human serum (HS). HS seemed to increase the number of ICC formed as compared to that obtained with FCS. A total of more than 100,000 ICC were produced from each of 3 litters, ages 67-77 days, after culture in the presence of HS. The DNA content of such ICC was reduced by about 50% as compared to those maintained with FCS supplementation. Immunocytochemical staining revealed insulin- and glucagon-positive cells scattered among a majority of nonstained cells within the cell clusters. ICC maintained in either FCS or HS displayed significant rates of (pro)insulin biosynthesis in vitro and an increased insulin release when exposed to 16.7 mM glucose plus 5 mM theophylline. Four weeks after implantation, ICC grafted under the kidney capsule of nondiabetic nude mice contained frequent insulin- and glucagon-positive cells. In 2 nude mice transplanted with ICC, the functional capacity of the graft was tested by perfusing the graft-bearing kidney. When the perfusion fluid was changed from one containing 2.8 mM glucose to one containing 16.7 mM glucose +/- 5 mM theophylline, the secretion of insulin increased within a few min. It is concluded that the fetal porcine pancreas can be used for large-scale production of ICC, which have a very consistent, but immature functional capacity. Because of their inherent growth and differentiation properties, fetal porcine ICC constitute a potential source of xenogenic islet grafts intended for human diabetics.