Islet Cell Aggregates Research Articles

203-LB: Redosing of Suboptimally Controlled Diabetic Mice with Human Neo-islets, 3D Aggregates of Mesenchymal Stromal and Pancreatic Islet Cells, Durably Restores Euglycemia: Relevance to the Treatment of Diabetic Humans

We reported that the i.p. administration of allogeneic Neo-Islets (NIs; 3D aggregates of allogeneic, culture-expanded Islet cells (ICs) with immune- and cyto-protective Mesenchymal Stromal Cells (MSCs)), durably restores normoglycemia in autoimmune type-1 diabetic NOD mice through omental engraftment. Similarly, our ongoing, FDA-guided pilot study in insulin dependent pet dogs (INAD 012-0776) demonstrates that allogeneic canine NI therapy significantly improves both glycemic control and insulin needs (> 1.5 years), and this without eliciting an allo-immune response. The current study was undertaken in preparation of a Phase I Clinical Trial in order to determine whether NIs engineered from human cells (hNIs) could also reduce insulin need, and whether redosing of suboptimally controlled diabetic mice could fully restore euglycemia, as is frequently required in human recipients of islet transplants. Hyperglycemia (blood glucose > 350 mg/dL x 3 days) was induced with streptozotocin in adult NOD/SCID mice and blood glucose levels were controlled with insulin pellets (Linbits) until i.p. hNI (2x10e5 hNIs/kg bw) or vehicle administration (n=6/group). Blood glucose levels, weights, ip glucose tolerance tests [GTT) were closely monitored. While no mice in the hNI treated group died (vs. 4 in the control group), hNI therapy partially reduced glucose levels . Therefore, on day 64 post initial treatment, mice were re-dosed with hNIs (2x10e5 hNIs/kg bw) and followed together with controls as above. This second dose of hNI completely and durably restored euglycemia and normalized ip GTTs. We conclude that this minimally invasive, safe and highly effective therapy supports our current pursuit of an IND for the conduct of a pioneering Clinical Trial in subjects with T1DM. Disclosure S.S. Chowdhury: Employee; Self; SymbioCellTech. A. Gooch: Employee; Self; SymbioCellTech. P. Zhang: Employee; Self; SymbioCellTech. Z. Hu: Employee; Self; SymbioCellTech. C. Westenfelder: Consultant; Self; SymbioCellTech.

Interim report on the effective intraperitoneal therapy of insulin-dependent diabetes mellitus in pet dogs using "Neo-Islets," aggregates of adipose stem and pancreatic islet cells (INAD 012-776).

We previously reported that allogeneic, intraperitoneally administered “Neo-Islets,” composed of cultured pancreatic islet cells co-aggregated with high numbers of immunoprotective and cytoprotective Adipose-derived Stem Cells, reestablished, through omental engraftment, redifferentiation and splenic and omental up-regulation of regulatory T-cells, normoglycemia in autoimmune Type-1 Diabetic Non-Obese Diabetic (NOD) mice without the use of immunosuppressive agents or encapsulation devices. Based on these observations, we are currently testing this Neo-Islet technology in an FDA guided pilot study (INAD 012–776) in insulin-dependent, spontaneously diabetic pet dogs by ultrasound-guided, intraperitoneal administration of 2x10e5 Neo-Islets/kilogram body weight to metabolically controlled (blood glucose, triglycerides, thyroid and adrenal functions) and sedated animals. We report here interim observations on the first 4 canine Neo-Islet-treated, insulin-dependent pet dogs that are now in the early to intermediate-term follow-up phase of the planned 3 year study (> 6 months post treatment). Current results from this translational study indicate that in dogs, Neo-Islets appear to engraft, redifferentiate and physiologically produce insulin, and are rejected by neither auto- nor allo-immune responses, as evidenced by (a) an absent IgG response to the allogeneic cells contained in the administered Neo-Islets, and (b) progressively improved glycemic control that achieves up to a 50% reduction in daily insulin needs paralleled by a statistically significant decrease in serum glucose concentrations. This is accomplished without the use of anti-rejection drugs or encapsulation devices. No adverse or serious adverse events related to the Neo-Islet administration have been observed to date. We conclude that this minimally invasive therapy has significant translational relevance to veterinary and clinical Type 1 diabetes mellitus by achieving complete and at this point partial glycemic control in two species, i.e., diabetic mice and dogs, respectively.

Size effect of engineered islets prepared using microfabricated wells on islet cell function and arrangement.

Pancreatic islets are heterogeneous clusters mainly composed of α and β cells, and these clusters range in diameter from 50 to several hundred micrometers. Native small islets are known to have a higher insulin secretion ability in vitro and to provide better transplantation outcomes when compared with large islets. In this study, we prepared microengineered pseudo-islets from dispersed rat islet cells using precisely-fabricated agarose gel-based microwells with different diameters (100, 300, or 500 μm) to investigate the function and survival of islet cell aggregates with well-controlled sizes. We observed that dead cells were rarely present in the small pseudo-islets with an average diameter of ∼60 μm prepared using 100 μm microwells. In contrast, we observed more dead cells in the larger pseudo-islets prepared using 300 and 500 μm microwells. The relative amount of hypoxic cells was significantly low in the small pseudo-islets whereas a hypoxic condition was present in the core region of the larger pseudo-islets. In addition, we found that the small-sized pseudo-islets reconstituted the in vivo-tissue like arrangement of the α and β cells, and restored the high insulin secretory capacity in response to high glucose. These results clearly suggest that precise size control of pseudo-islets is essential for maintaining islet cell function and survival in vitro. The small-sized pseudo-islets may be advantageous for providing a better therapeutic approach for treating type 1 diabetes mellitus via islet reorganization and transplantation.

Transplantation of Co-aggregates of Sertoli Cells and Islet Cells Into Liver Without Immunosuppression

Transplantation of islets of Langerhans (islets) was used to treat insulin-dependent diabetes mellitus. However, islet grafts must be maintained by administration of immunosuppressive drugs, which can lead to complications in the long term. An approach that avoids immunosuppressive drug use is desirable. Co-aggregates of Sertoli cells and islet cells from BALB/c mice that were prepared by the hanging drop method were transplanted into C57BL/6 mouse liver through the portal vein as in human clinical islet transplantation. The core part of the aggregates contained mainly Sertoli cells, and these cells were surrounded by islet cells. The co-aggregates retained the functions of both Sertoli and islet cells. When 800 co-aggregates were transplanted into seven C57BL/6 mice via the portal vein, six of seven recipient mice demonstrated quasi-normoglycemia for more than 100 days. The hanging drop method is suitable for preparing aggregates of Sertoli and islet cells for transplantation. Notably, transplantation of these allogeneic co-aggregates into mice with chemically induced diabetes via the portal vein resulted in long-term graft survival without systemic immunosuppression.

Proliferation and Functional Assessment of Pseudo-islets With the Use of Pancreatic Endocrine Cells

Many obstacles beset islet transplantation, particularly insufficient tissue mass. Previously, we reported production of pseudo-islets. In addition, there have been reports in which coculture with pancreatic islet and bone marrow mesenchymal stem cells (BMSCs) demonstrated positive effects on pancreatic islet function. The purpose of this study was to perform morphologic and functional evaluations of pancreatic pseudo-islets cocultured with BMSCs. Pancreatic endocrine cells (PECs) were collected with a previously reported method; bone marrow was aspirated from the rat femur. Subsequently, PECs and BMSCs cocultured at high density on low-cell-binding culture dishes kept suspended by shaking. The functionality and characteristics of the mixed cell complexes were evaluated by glucose challenge, insulin enzyme-linked immunosorbent assay, reverse-transcription polymerase chain reaction, and immunohistochemistry. Through expansion for 2 weeks in continuous culture passages, ∼1 million PECs were recovered after aggregation. They presented spherical shapes and sizes similar to naïve islets, according to phase-contrast microscopy. The spheroid aggregates of pancreatic islet cells and BMSCs showed fortified functions and maintained viability. In conclusion, PECs served as a cell source for pseudo-islets, which were both morphologically and genetically similar to naïve islets. We also suggest a manufacturing method for mixed cellular complexes from 2 different origins that can improve secretion ability and cell differentiation.

Molecular basis for the regulation of islet beta cell mass in mice: the role of E-cadherin.

In rodents and humans, the rate of beta cell proliferation declines rapidly after birth; formation of the islets of Langerhans begins perinatally and continues after birth. Here, we tested the hypothesis that increasing levels of E-cadherin during islet formation mediate the decline in beta cell proliferation rate by contributing to a reduction of nuclear β-catenin and D-cyclins. We examined E-cadherin, nuclear β-catenin, and D-cyclin levels, as well as cell proliferation during in vitro and in vivo formation of islet cell aggregates, using β-TC6 cells and transgenic mice with green fluorescent protein (GFP)-labelled beta cells, respectively. We tested the role of E-cadherin using antisense-mediated reductions of E-cadherin in β-TC6 cells, and mice segregating for a beta cell-specific E-cadherin knockout (Ecad [also known as Cdh1] βKO). In vitro, pseudo-islets of β-TC6 cells displayed increased E-cadherin but decreased nuclear β-catenin and cyclin D2, and reduced rates of cell proliferation, compared with monolayers. Antisense knockdown of E-cadherin increased cell proliferation and levels of cyclins D1 and D2. After birth, beta cells showed increased levels of E-cadherin, but decreased levels of D-cyclin, whereas islets of Ecad βKO mice showed increased levels of D-cyclins and nuclear β-catenin, as well as increased beta cell proliferation. These islets were significantly larger than those of control mice and displayed reduced levels of connexin 36. These changes correlated with reduced insulin response to ambient glucose, both in vitro and in vivo. The findings support our hypothesis by indicating an important role of E-cadherin in the control of beta cell mass and function.

Islets Transplanted in Immunoisolation Devices: A Review of the Progress and the Challenges that Remain

The concept of using an immunoisolation device to facilitate the transplantation of islets without the need for immunosuppression has been around for more than 50 yr. Significant progress has been made in developing suitable materials that satisfy the need for biocompatibility, durability, and permselectivity. However, the search is ongoing for a device that allows sufficient oxygen transfer while maintaining a barrier to immune cells and preventing rejection of the transplanted tissue. Separating the islets from the rich blood supply in the native pancreas takes its toll. The immunoisolated islets commonly suffer from hypoxia and necrosis, which in turn triggers a host immune response. Efforts have been made to improve the supply of nutrients by using proangiogenic factors to augment the development of a vascular supply in the transplant site, by using small islet cell aggregates to reduce the barrier to diffusion of oxygen, or by creating scaffolds that are in close proximity to a vascular network such as the omental blood supply. Even if these efforts are successful, the shortage of donor islet tissue available for transplantation remains a major problem. To this end, a search for a renewable source of insulin-producing cells is ongoing; whether these will come from adult or embryonic stem cells or xenogeneic sources remains to be seen. Herein we will review the above issues and chart the progress made with various immunoisolation devices in small and large animal models and the small number of clinical trials carried out to date.

Rat islet cell aggregates are superior to islets for transplantation in microcapsules

Islet transplantation is a promising treatment for type 1 diabetes but is hampered by a shortage of donor human tissue and early failure. Research on islet cell transplantation includes finding new sources of cells and immunoisolation to protect from immune assault and tumourigenic potential. Small islet cell aggregates were studied to determine if their survival and function were superior to intact islets within microcapsules because of reduced oxygen transport limitation and inflammatory mediators. Islet cell aggregates were generated by dispersing rat islets into single cells and allowing them to re-aggregate in culture. Rat islets and islet cell aggregates were encapsulated in barium alginate capsules and studied when cultured in low (0.5% or 2%) or normal (20%) oxygen, or transplanted into mice. Encapsulated islet cell aggregates were able to survive and function better than intact islets in terms of oxygen-consumption rate, nuclei counts, insulin-to-DNA ratio and glucose-stimulated insulin secretion. They also had reduced expression of pro-inflammatory genes. Islet cell aggregates showed reduced tissue necrosis in an immunodeficient transplant model and a much greater proportion of diabetic xenogeneic transplant recipients receiving islet cell aggregates (tissue volume of only 85 islet equivalents) had reversal of hyperglycaemia than recipients receiving intact islets. These aggregates were superior to intact islets in terms of survival and function in low-oxygen culture and during transplantation and are likely to provide more efficient utilisation of islet tissue, a finding of importance for the future of cell therapy for diabetes.

Oxygen consumption and diffusion in assemblages of respiring spheres: Performance enhancement of a bioartificial pancreas

Abstract Microencapsulation provides an immune barrier for transplanted islets of Langerhans to treat diabetes, but it imposes oxygen diffusional limitations that can result in loss of viability and function. We investigate two methods to reduce oxygen transport limitations: (1) enhancement of the encapsulant oxygen permeability, for example, by combination of a highly concentrated perfluorocarbon (PFC) emulsion with alginate (PFC alginate); and (2) reduction of islet tissue size, for example, by dispersing the islets into single cells followed by reaggregation into cell clusters smaller than the original islet. A theoretical reaction–diffusion model is used to predict the three-dimensional distribution of oxygen partial pressure in a spherical microcapsule and a planar slab containing islet tissue, from which the loss of cell viability and the reduction in insulin secretion rate is estimated. Numerical simulations are carried out for normal alginate and PFC alginate to examine the effect of surface oxygen partial pressure, capsule diameter, slab thickness, and the size and density of dispersed islet tissue. Results show that hypoxic conditions can be reduced, thereby enhancing islet viability and substantially maintaining insulin secretion rate when PFC emulsion is incorporated in the encapsulation material or when smaller islet cell aggregates are used in both types of geometries.

Hypoglycemia after Pancreas Transplantation: Association with Allograft Nesidiodysplasia and Expression of Islet Neogenesis-Associated Peptide

We report a case of severe hypoglycemia occurring in a 35-yr-old woman, 6 yr after pancreas transplantation for type 1 diabetes mellitus. Extensive preoperative and intraoperative exploration failed to disclose the presence of a focal adenomatous lesion. Partial allograft pancreatectomy was performed initially, but it failed to control the hypoglycemic symptoms, leading to complete removal of the pancreas allograft. Histopathological examination of the resected pancreas allograft showed the presence of nesidioblastosis, characterized by foci of islet cells budding off ducts, accompanied by an increase in the number of islets, numerous small intralobular islet cell aggregates, and nesidiodysplasia (large, hyperchromatic islet cell nuclei). Islet neogenesis-associated protein-positive islets and ducts were seen by immunofluorescence. Insulin-positive islets ranged from very small to large, with isolated insulin-positive cells diffusely scattered, consistent with islet neogenesis. Very little glucagon staining was identified. Reported cases of adult nesidioblastosis are reviewed. The significance of nesidioblastosis in the context of pancreas transplantation and possible mechanisms of posttransplant hypoglycemia are discussed.

Formation of Sertoli cell-enriched tissue constructs utilizing simulated microgravity technology.

Cell transplantation therapy for diabetes and Parkinson's disease offers hope for long-term alleviation of symptoms. However, successful protocols remain elusive due to obstacles, including rejection and lack of tropic support for the graft. To enhance engraftment, testis-derived postmitotic Sertoli cells have been cotransplanted with islets in the diabetic rat (Db) and neurons in the Parkinsonian rat (PD). Sertoli cell tropic, regulatory, and nutritive factors that nourish and stimulate germ cells also support isolated neurons and islets in vitro. Likewise, immunosuppressive properties of Sertoli cells, extant in the testis, are expressed by extratesticular Sertoli cells evidenced by allo- and xenograft immunoprotection of grafts in both the CNS (in the PD model) and the periphery (in the Db model). On this basis, we have created Sertoli islet cell aggregates (SICA) and Sertoli neuron aggregated cells (SNAC) using simulated microgravity culture technology developed by NASA. Isolated rat and pig Sertoli cells were cocultured with neonatal pig islets (SICA) and with immortalized N-Terra-2 (NT2) neurons (SNAC) in the HARV biochamber. Formed aggregates were assayed for desirable functional and structural characteristics. Cell viability in SICA and SNAC exceeded 90% and FasL immunopositive Sertoli cells were present in both. Sertoli cells did not interfere with insulin secretion by SICA and promoted differentiation of NT2 cells to the dopaminergic hNT cell type in SNAC. Addition of Matrigel resulted in structural reorganization of the aggregates and enhanced insulin secretion. We conclude that SICA, SNAC, and Matrigel-induced islet- and neuron-filled "Sertoli cell biochambers" are suitable for long-term transplantation treatment of Db and PD.

Potential application of neonatal porcine islets as treatment for type 1 diabetes: a review.

Islet transplantation has been shown to be a viable option for treating patients with type 1 diabetes. However, widespread clinical application of this treatment will necessitate an alternative source of insulin-producing tissue. Porcine pancreata may be a potential source of islets since pigs are inexpensive, readily available, and exhibit morphological and physiological characteristics comparable to humans. Recently, we developed a simple, standardized procedure for isolating large numbers of neonatal porcine islets with a reproducible and defined cellular composition. Following nine days of in vitro culture, tissue from one neonatal pig pancreas yielded approximately 50,000 islet cell aggregates, consisting of primarily epithelial cells (57%) and pancreatic endocrine cells (35%). In addition, neonatal porcine islets were responsive to glucose challenge in vitro and were capable of correcting hyperglycemia in alloxan-induced diabetic nude mice. Although neonatal porcine islets constitute an attractive alternative source of insulin-producing tissue for clinical transplantation, many aspects such as the immunological responses to these tissue and the latent period (2 to 8 weeks) between transplantation of these islets and the reversal of hyperglycemia need further investigation. This article discusses these issues and presents possible solutions to problems that may hinder the potential application of neonatal porcine islets for transplantation into patients with type 1 diabetes.

Expression of Gal alpha(1,3)gal on neonatal porcine islet beta-cells and susceptibility to human antibody/complement lysis.

Neonatal porcine pancreases may be a potential source of islets for transplantation into patients with type 1 diabetes; however, whether these cellular grafts will be susceptible to damage by human natural antibody-mediated rejection remains controversial. Although we and others have demonstrated that porcine islets bind human IgG and IgM, it remains unknown if they express the xenoreactive antigen Gal alpha(1,3)Gal beta(1,4)GlcNAc-R (Gal epitope). In this study, by using the Gal-specific lectin IB4 for immunohistochemistry and fluorescence-activated cell sorter (FACS) analysis, we determined which cell types present in porcine neonatal islet cell (NIC) aggregates express the Gal epitope and which ones are susceptible to lysis by activation of the human complement. After FACS analysis, 30.0 +/- 3.0% of porcine NICs were shown to express Gal, whereas 70.0 +/- 2.0% did not. Histological assessment of Gal-expressing cells revealed that 54.9 +/- 8.8% stained positive for either insulin or glucagon. In contrast, 68.8 +/- 8.4% of the Gal-negative population stained positive for the pancreatic hormones insulin and glucagon. Incubation of either the Gal-positive or -negative cells with human AB serum plus complement for 1.5 h resulted in the lysis of >90% of the cells. These results demonstrate that porcine NIC aggregates are composed of Gal-expressing cells and that expression of Gal is not restricted to nonendocrine cells. Furthermore, both Gal-positive and Gal-negative cells are susceptible to human antibody/complement-mediated cytolysis, suggesting that this form of immunological destruction is an obstacle that will need to be overcome before porcine NIC aggregates can be used clinically.

Oscillatory Ca 2+ signaling in somatostatin-producing cells from the human pancreas

Oscillatory Ca 2+ signaling was studied in human somatostatin-releasing pancreatic δ cells identified by immunostaining. A ratiometric fura-2 technique was used for measuring cytoplasmic concentrations of Ca 2+ and Sr 2+ in δ cells exposed to the respective cation. Rhythmic activity in terms of slow (frequency, 0.1 to 0.4 per minute) oscillations from close to the basal level was seen in the presence of 3 to 20 mmol/L glucose during superfusion with medium containing 2.6 to 5 mmol/L Ca 2+ or 5 mmol/L Sr 2. These oscillations could be transformed into a sustained increase by decreasing extracellular Ca 2+ or adding 1 mmol/L tolbutamide or 20 nmol/L glucagon. Addition of glucagon to a medium containing 20 mmol/L glucose resulted in the generation of short (< 30 seconds) transients, which disappeared upon exposure to 100 nmol/L of the intracellular Ca 2+-adenosine triphosphatase (ATPase) inhibitor thapsigargin. When analyzing small aggregates of islet cells, it became evident that oscillatory activity in δ cells can be synchronous with that in adjacent non—δ cells. It is concluded that secretion of pancreatic somatostatin in man involves Ca 2+ signaling similar to that regulating the pulsatile release of insulin.

Glucose Induces Cytoplasmic Na+Oscillations in Pancreatic β-Cells

The cytoplasmic Na+concentration ([Na+]i) was measured in individual mouse β-cells and islet cell aggregates using the indicator SBFI. Small oscillations of [Na+]iwere occasionally observed after raising glucose from 3 to 11 mM. Distinct oscillations with a frequency of 0.29 ± 0.02 min−1(n=18) and amplitudes of 7.4 ± 0.5 mM (n=18) were obtained when also adding the Na+channel agonist veratridine. The oscillations were counteracted by tetrodotoxin or by lowering the glucose concentration to 3 mM. Glucose failed to induce oscillations when increasing Na+entry by co-transport with L-glycine or raising [Na+]iby blocking the Na/K pump with ouabain. The results indicate that glucose can induce oscillations of [Na+]iin pancreatic β-cells, although this effect is usually less obvious due to activation of an efficient Na/K pump.

Large scale isolation, growth, and function of porcine neonatal islet cells.

Based upon existing methods of isolating fetal porcine islet tissue, a simple, reliable procedure was developed for the preparation of porcine neonatal islet cell aggregates with a reproducible and defined cellular composition. After 9 d of in vitro culture, tissue from one neonatal pig pancreas yielded approximately 50,000 islet cell aggregates, consisting of primarily epithelial cells (57%) and pancreatic endocrine cells (35%). During the culture period, the total beta cell mass decreased initially, but subsequently increased 1.5-fold between days 3 and 9. Transplantation of grafts consisting of 3 x 10(5) beta cells (1,000 aggregated) under the kidney capsule of alloxan-diabetic nude mice corrected hyperglycemia in 75% (10/13) of the animals, whereas, 100% (20/20) of recipients implanted with 6 x 10(5) beta cells (2,000 aggregates) achieved euglycemia within 8 wk posttransplantation. Nephrectomy of the graft bearing kidney at 14 wk posttransplantation resulted in hyperglycemia in all recipients, and examination of the grafts revealed the presence of numerous well-granulated insulin- and glucagon-containing cells. The cellular insulin content of these grafts was 20 to 30-fold higher than at the time of transplantation. These results indicate that the neonatal porcine pancrease can be used as a source of large numbers of viable islet cells, which have the potential for growth both in vitro and in vivo, and exhibit the metabolic capacity to correct diabetes in nude mice.

Neonatal pig pancreata as a source of insulin‐producing tissue for xenotransplantation

Abstract: Islet transplantation has been proven as a viable option for treating type 1 diabetics; however, widespread clinical application of this technique will necessitate an alternative source of insulin‐producing tissue. Porcine pancreata may be a potential source of islets, but adult porcine islets are difficult to isolate, are fragile, and have a tendency to fall apart during culture. In this study, we investigated the feasibility of preparing functionally viable islet cells in large quantities from the neonatal pig pancreas. Following collagenase digestion and 7 days of tissue culture, a mean of 48,000 neonatal islet cell (NIC) aggregates were recovered per pancreas. These NICs were responsive to glucose in vitro and were capable of correcting hyperglycemia in alloxan‐induced diabetic nude mice. These results indicate that the neonatal porcine pancreas can be used to produce large numbers of functionally viable islet cells.

Transplantation of Purified Islet Cells in Diabetic Rats: III. Immunosuppressive Effect of Cyclosporin

This study examined the effect of cyclosporin on the survival of islet beta-cell allografts in streptozocin (STZ)-induced diabetic rats. At a daily oral dose of 5 mg/kg, the agent prevented the rejection of isolated islets, provided they were little contaminated by other pancreatic tissue. The immunosuppressive effect rapidly disappeared after discontinuation of the drug, except when the donor tissue had been pretreated to reduce its nonendocrine content. All recipients of cultured and selected islets maintained a normalized state for greater than 15 wk beyond the 5-wk drug course; this was not the case for shorter periods of treatment. A long-term beneficial effect was also observed in all recipients of purified islet beta-cell grafts, which reversed without treatment in half of the cases. Cyclosporin markedly reduced the mononuclear cell infiltration in each type of islet beta-cell allograft; aggregates of mixed endocrine islet cells were kept virtually infiltration free. Conditions with minimal initial infiltration were associated with long-term graft survival without the need for continuous pharmacological immunosuppression. We conclude that a short-term cyclosporin treatment can induce long-term survival of allografted islet beta-cells, provided the grafts are only slightly contaminated by nonendocrine elements. In rodents, sufficient immunosuppression was achieved by circulating cyclosporin levels of 100-400 ng/ml. Higher concentrations were cytotoxic for cultured islet beta-cells and islet non-beta-cells. A 5-wk treatment with the immunomodulator ciamexone also resulted in long-term survival of purified beta-cell allografts but not of cultured islets.(ABSTRACT TRUNCATED AT 250 WORDS)

Transplantation of Purified Islet Cells in Diabetic Rats: II. Immunogenicity of Allografted Islet β-Cells

This study examines whether the survival of allografted rat islet beta-cells is influenced by the presence of other pancreatic donor cells. Grafts (RT1u/l) of different cellular composition were intraportally transplanted in streptozocin-induced diabetic rats (RT1n/n). All grafts corrected the diabetic state within 3 days. Implants of freshly isolated islets contained various endocrine and nonendocrine cell types; they became diffusely infiltrated within 1 wk and were completely destroyed within 2 wk. A 4-day culture period did not lead to major changes in the cellular composition of the islets or in their survival as allograft. Islet cell aggregates prepared after islet dissociation and cell purification were less acutely infiltrated and less rapidly rejected. Aggregates composed of sorted MHC class II-negative cells maintained basal normoglycemia in 3 of 5 recipients for 5 wk but only in 1 of 5 for 20 wk. Aggregates of purified islet beta-cells remained relatively free of diffuse infiltrations during the 1st wk and preserved the normalized state in 7 of 13 recipients for 5 wk; after 20 wk, 6 of 13 were still aglucosuric, but 40% of the implants were diffusely infiltrated and depleted of insulin. Reaggregation of purified islet beta-cells with purified islet endocrine non-beta-cells promoted their long-term survival as allograft: 11 of 13 recipients of mixed islet endocrine cells maintained normal basal glycemia over 20 wk; their implants contained relatively constant insulin reserves and remained virtually devoid of diffuse infiltrations. These results demonstrate that techniques aiming at the elimination of surface MHC class II-positive cells are less successful in preparing rat islet allografts of low immunogenicity than methods of positive cell selection. Pure islet beta-cells are immunogenic as an allograft but illicit a milder and less-acute immune attack than undissociated islet tissue. Nonendocrine and damaged islet cells are suspected of enhancing the rapidity and intensity of the cytotoxic reaction. Survival of allografted beta-cells is markedly prolonged by the presence of islet endocrine non-beta-cells within the graft. The mechanisms underlying this effect have not yet been elucidated; they may involve immune and metabolic interactions of the endocrine non-beta-cells. We conclude that purification of islet endocrine cells represents a new and powerful method for preparing insulin-producing allografts that can survive in hosts without pharmacological immunosuppression.

Islet-cell aggregates: Structure and insulin-producing activity during in vitro culture

Islet-cell aggregates: Structure and insulin-producing activity during in vitro culture