Angiogenesis and angioarchitecture of transplanted fetal porcine islet-like cell clusters

Normoglycemic, athymic nude mice were implanted with 3 microl (approximately 250) fetal, porcine islet-like cell clusters under the renal capsule. The angioarchitecture of the transplanted islets was studied by microvascular corrosion casts 3 or 52 weeks after implantation. Arterioles were few, and observed mainly in the older age group. This is likely to be due to the fact that the arterioles were derived from intrarenal blood vessels, i.e., they were not visible on the graft surface. Within the grafts nests of capillaries, probably supplying a single islet-like cell clusters, could be seen in both groups. Numerous capillary sprouts were seen within the graft after 3 weeks, and to a slighter extent also after 1 year. Moreover, especially in grafts examined 3 weeks, but also 52 weeks, after transplantation, holes were observed in dilated capillary segments, suggesting that intussusceptive microvascular growth occurred in parallel with angiogenesis. A well-developed microvasculature could be observed 52 weeks after transplantation, whereas the number of capillaries in the implant was less pronounced 3 weeks postimplantation. The efferent venules were located peripherally in the islets and drained immediately into larger veins, derived from capsular veins clearly seen on the surface of the graft. It is concluded that xenotransplanted islet-like cell clusters develop an autonomous microcirculation by stimulating angiogenesis from surrounding blood vessels. Our findings suggest that single islet-like cell clusters remain morphologically intact after transplantation, and probably function as single endocrine units rather than forming a single homogenous endocrine tissue. Furthermore, it seems as if a continuous reorganization of the vasculature, with an associated angiogenesis, occurs throughout the observation period.     

微囊化新生猪胰岛样细胞团异种移植

本实验研究新生猪与大鼠产的异种移植。结果显示，新生猪胰岛样细胞团可纠正糖尿病大鼠的高血糖状态；海灌酸钠－多聚赖氨酸微囊能有效保护移植物的存活，平均存活时间为４个月。而微囊作为一种新型防排斥手段，将应用于广泛的领域。     

Functional and immunohistochemical evaluation of porcine neonatal islet-like cell clusters

Porcine neonatal islet-like cell clusters (NICCs) may be an attractive source of insulin-producing tissue for xenotransplantation in type I diabetic patients. We examined the functional and immunohistochemical outcome of the islet grafts in vitro during long-term culture and in vivo after transplantation to athymic nude mice. On average we obtained 29,000 NICCs from each pancreas. In a perifusion system, NICCs responded poorly to a glucose challenge alone, but 10 mmol/L arginine elicited a fourfold increase in insulin secretion and 16.7 mmol/L glucose + 10 mmol/L arginine caused a sevenfold increase in insulin section indicating some sensitivity towards glucose. Hormone content as well as the number of hormone-containing cells increased for the first 14 days of culture. When NICCs were stained for hormones, proliferation (Ki67), and duct cells (CK7), some insulin- and glucagon-positive cells co-stained for proliferation. However no co-staining was observed between insulin- and glucagon-positive cells or between hormone-and CK-positive cells. Following transplantation of 2000 NICCs under the renal capsule of diabetic nude mice, BG levels were normalized within an average of 13 weeks. Oral and IP glucose tolerance tests revealed a normal or even faster clearance of a glucose load compared with normal controls. Immunohistochemical examination of the grafts revealed primarily insulin-positive cells. In summary, in vitro, NICCs responded to a challenge including glucose and arginine. There was a potential for expansion of the beta-cell mass of NICCs in vitro as well as in vivo where NICCs eventually may normalize blood glucose of diabetic mice.     

Fetal porcine islet-like cell clusters transplanted to cynomolgus monkeys: an immunohistochemical study

BACKGROUND: The mechanism(s) involved in acute cellular xenograft rejection have hitherto been generated in vitro or in different experimental models, with pig tissue being transplanted to rodents. There is an urgent need to validate these results in a clinically more relevant combination of species. METHODS: Fetal porcine islet-like cell clusters (ICC) were transplanted under the kidney capsule in cynomolgus monkeys, either untreated or given immunosuppression with cyclosporine (CsA; 10 mg/kg body weight, intramuscularly) and 15-deoxyspergualin (DSG; 5 mg/kg body weight, intramuscularly). ICC xenografts were examined at 1, 3, 6, or 10-12 days after transplantation, using immunohistochemical techniques. Serum levels of xenoreactive antibodies were measured with ELISA. RESULTS: No deposits of IgM, IgG, Clq, or C3 were detected within the ICC xenograft in any of the monkeys. Likewise, no significant increase in the levels of xenoreactive antibodies were found after transplantation. In untreated animals, a few N-Elastase-positive cells (neutrophil granulocytes) were seen in the xenograft at day 1. A few mononuclear cells were present in the adjacent renal parenchyma, but they did not infiltrate the xenograft. At this time (day 1), early signs of necrosis were observed in the central parts of the graft. On day 3, the graft had a large, central necrotic area that contained polymorphonuclear cells; the remaining parts of the xenograft showed severe infiltration with CD8+ T cells. Occasional CD68+ cells (macrophages) were seen on days 1 and 3. On day 6, large numbers of macrophages were found infiltrating the entire graft. A few CD20+ B cells, accumulated as small clusters, were also found. Only a few natural killer cells (CD56+) were detected. The CsA/DSG-treated monkeys showed markedly fewer CD2+/CD8+ T cells on day 6 than the untreated monkeys, and the ICC graft was clearly better preserved. However, the number of CD8+ and CD68+ cells had increased considerably at 12 days after transplantation and diffusely infiltrated the whole ICC xenograft. CONCLUSION: Porcine ICC transplanted under the kidney capsule in cynomolgus monkeys were rejected by an acute cell-mediated rejection progressing during the first 6 days after transplantation. The process was not dependent on host Ig or C3 binding to the graft. Although the rejection of porcine ICC was significantly delayed in CsA/DSG-treated monkeys, the ICC xenografts were almost completely destroyed 12 days after transplantation.     

Microbiological characterization of porcine fetal islet-like cell clusters for intended clinical xenografting

Abstract: A major concern in animal-to-man transplantation is the risk of transferring microorganisms from an animal to a patient. For this reason, before transplanting the porcine fetal pancreas to diabetic patients, the pregnant sows and the tissue to be transplanted—i.e., fetal islet-like cell clusters (ICC) prepared by collagenase digestion and cell culture—were subjected to a comprehensive microbiological screening program. Serological testing of the pregnant sows revealed antibodies to Leptospira interrogans (2/59 sows, 3%) and Aspergillus fumigatus (23/41, 56%). However, there was no evidence of colonization of the animals with these organisms. Serological testing for a multitude of other microorganisms was negative. Growth of bacteria was found in 4% of the specimens recovered from the endometrium and in 31% of those from the amniotic fluid. However, none of these bacterial species was recovered in the culture dishes containing ICC. A few samples taken from the culture dishes (2/53, 4%) revealed growth of bacteria, but no viruses, fungi, or mycoplasmas were detected. In nude mice that had carried ICC under the kidney capsule for 9 to 23 months, various lesions were observed, but none of them was due to the implanted porcine tissue. Microscopic examination of the mouse brains revealed no prion-related alterations. If porcine tissue is to be transplanted into humans, a stringent microbiological screening program must be followed. The program used by us makes possible the weeding out of seropositive animals, as required, and the exclusion of contaminated tissue.     

Effects of micro-encapsulation on morphology and endocrine function of cryopreserved neonatal porcine islet-like cell clusters

BACKGROUND: For the success of clinical islets transplantation, the development of a long-term storage method is necessary. However, the structure of digested islets is scanty for culture and cryopreservation. In this study, the effect of micro-encapsulation to cryopreserved porcine islet-like cell clusters (ICCs) was investigated. METHODS: The ICCs prepared from neonatal pigs by collagenase digestion and culture technique were cryopreserved and micro-encapsulated in 5% agarose membranes. After cryopreservation, ICC cultured without encapsulation (group A) and cultured with encapsulation (group B) were assessed by comparison with no cryopreserved ICC (control) both in vitro by static incubation test and in vivo in a xenotransplantation study. RESULTS: Micro-encapsulation was able to maintain the fine morphology and the number of ICCs of group B after 7 days of culture. There were not significant differences in insulin secretion of group B and control on day 1 and 7 of culture (1 day:11+/-0.99, 7 days: 5.30+/-1.08 microU/ICC/hr NS versus control). On day 7 of culture, the retrieval rate of group B (105.2+/-9.8%) is obviously higher compared with group A (63.0+/-6.3%). In the xenotransplatation model, the ICCs of group B showed long survival time (7.9+/-0.4 weeks) and good transplantation effect. CONCLUSION: Our study suggests that micro-encapsulation is one of the useful method for cryopreserved ICC to maintain the fine morphology and effectively recover the endocrine function.     

Xenograft rejection of fetal porcine islet-like cell clusters in the rat: effects of active and passive immunization

The process of islet xenograft rejection is still poorly understood. To elucidate further possible mechanism(s) involved in xenograft rejection, the effect of different immunization protocols was investigated. Fetal porcine islet-like cell clusters (ICCs) were transplanted under the kidney capsule in otherwise untreated rats, rats pre-immunized by s.c. injections of ICCs and in rats passively immunized with immune serum. The rejection process was evaluated with regard to antibody and complement deposition in the graft, as well as to morphology and phenotype of the infiltrating cells. In otherwise untreated animals, a moderate perigraft mononuclear cell infiltrate was seen after 3 days. Graft destruction became evident on day 6 with marked intragraft infiltration by macrophages (ED1 positive), whereas T cells were in the minority and mainly located in the perigraft area. In contrast to the findings in non-immunized rats, the rejection process in pre-immunized rats was characterized by marked intragraft infiltration by macrophages 3 days after transplantation. Moreover, both T cells and macrophages heavily infiltrated the adjacent kidney parenchyma, and major histocompatibility complex (MHC) class II expression in surrounding kidney tubular cells was concomitantly enhanced. Syngeneic rat islets mixed with porcine ICCs escaped the rejection process in non-immunized rats but were affected in pre-immunized animals. Thus, the specificity of the rejection process in non-immunized animals seems to be lost in pre-immunized animals. The early macrophage infiltration was also accelerated in rats passively immunized with immune serum, but no early switch from perigraft to intragraft infiltration or subsequent cellular infiltration in the adjacent kidney parenchyma was seen. Circulating xenoreactive antibodies of the IgG isotype increased after transplantation in normal and otherwise untreated rats. No distinct IgG deposition in the ICC xenografts was observed until day 12 after transplantation in untreated rats, whereas perigraft deposition of IgG was found 1 day after transplantation in pre-immunized rats and in rats given immune serum. No deposition of complement was observed within the ICC xenograft in any of the groups during the observation period. The dependence on T cells, the massive infiltration of macrophages with a unique phenotype, the cellular distribution, and the loss of specificity (bystander killing) of the rejection process in immunized rats suggest that ICC xenograft rejection shares some of its main characteristics with a delayed type hypersensitivity-like (DTH) immune response.     

A study of the glycoantigens of neonatal porcine islet-like cell clusters using a lectin microarray

Background

The pig pancreas is considered to be the most suitable source of islets for clinical xenotransplantation. Two types of islet transplantation are: adult pig islets and neonatal porcine islet-like cell clusters (NPCC). However, besides a-Gal expression, differences in glycosylation and xenoantigenicity between both types were not clear so fat to date. In this study, we performed lectin microarray analyses of NPCCs cultured for 1, 5, or 9 days.

Methods

We studied differences in gycoantigens among several kinds of wild-type NPCCs isolated from 1- to 3-day-old neonatal wild-type pigs (Large White/Landrace × Duroc) and cultured for 1, 5 and 9 days in Ham's 10 in the presence of nicotinamide, using a previously published technique. After sonication and centrifugation, supernatant proteins from each islet were labeled with Cy3, applied to a lectin array and scanned with an SC-Profiler for evaluation using an Array Pro Analyzer.

Results

The overall signals of NPCC at days 5 and 9, showed almost the same values to most lectins, whereas those on day 1 showed differences, suggesting that the NPCC on day 1 contain immature cells that gradually turn to mature NPCCs in culture.     

Comparison of fetal porcine aggregates of purified beta-cells versus islet-like cell clusters as a treatment of diabetes

Fetal pig islet-like cell clusters (ICCs) have the potential to reverse diabetes 1-5 months after transplantation. In a fetal ICC, however, beta-cells constitute only 6-8% of the cells, in contrast to 65% in an adult pig islet. Attempts to purify fetal beta-cells from cell clusters and compare their function to that of ICCs have not been shown previously. Beta-cells were purified from ICCs isolated from the fetal pig pancreas. These were then aggregated and maintained in culture for 3 days. ICCs were isolated from fetal pig pancreas and allowed to round up in culture for 3 days. Transplantation of aggregates and ICCs (10,000 and 12,600, respectively) into diabetic immunoincompetent mice resulted in normoglycemia at 18 +/- 2 and 8 +/- 1 weeks, respectively (p = 0.0006). Removal of grafts after normalization of blood glucose levels resulted in rapid return of hyperglycemia in both groups. In conclusion, a purified population of immature beta-cells can be produced from the fetal pig pancreas. The reason these cells take longer than ICCs to reverse diabetes when transplanted is postulated to be because of the relative lack of precursor cells from which beta-cells differentiate. This finding may have implications for stem cell therapy, as other cell types, other than purified beta-cells, may be necessary for appropriate function in vivo.     

Immunomodulation of fetal pig islet-like cell clusters by gamma-irradiation

Pretreatment of tissues to reduce their immunogenicity is an attractive option, and exposure of donor islets to gamma-irradiation has previously been shown to result in their prolonged survival when transplanted into rodents. Fetal pig islet-like cell clusters (ICCs) are currently under trial as a potential xenogeneic tissue for the treatment of type 1 diabetes. The purpose of this study was to examine in vivo and in vitro the immunomodulatory effects of gamma-irradiation on ICCs in a xenogeneic situation. The immunogenicity of gamma-irradiated ICCs was determined in a mixed islet lymphocyte culture (MILC), in which fetal pigs ICCs were able to stimulate human peripheral blood mononuclear cells (PBMCs). Exposure of the ICCs to gamma-irradiation significantly reduced their ability to stimulate PBMCs in a MILC when 10 Gy but not lower doses of irradiation were applied. However, this effect of gamma-irradiation was variable and was present only in those experiments in which the stimulation index was relatively low. Gamma-irradiation was toxic to ICCs in vitro, causing a reduction in the [³H]-thymidine incorporation of 82–94% at 5–20 Gy. This toxic effect of gamma-irradiation was also demonstrated in vivo: the insulin content of ICCs beneath the renal capsule in SCID mice treated with 5–20 Gy significantly was reduced (P < 0.05) 6 weeks after transplantation. Exposure of ICCs to gamma-irradiation (2.5 Gy) alone in vitro or in combination with injection of cyclosporine (12.5 mg/kg per day) did not prevent the rejection of ICCs transplanted beneath the renal capsule of BALB/c mice. We conclude that gamma-irradiation is toxic to fetal pig ICCs at a higher dose and at a lower dose, alone or in combination with cyclosporine, and was unable to prolong discordant islet xenograft survival in mice.     

Production and characterization of fetal sheep pancreatic islet-like cell clusters

Explants of fetal sheep pancreas transplanted into diabetic athymic mice survive for many months but there is only partial differentiation of the endocrine cells. As an alternative form of graft we examined the possibility of creating islet-like cell clusters (ICCs) by collagenase digestion of the fetal sheep pancreas, as has been described for human and porcine fetal pancreas. Such ICCs did form at the rate of 6–23 per 10 mg pancreas; their size varied between 65 and 474 μm (median 232 μm) and their insulin content was 1.6 ± 0.2 mU per 20 ICCs. Laser scanning confocal analysis showed that 4.6 ∓ 0.7% of the cells contained insulin. Insulin was secreted from ICCs maintained in culture at the daily rate of 2.5 mU per 30 ICCs. Arginine but not glucose or theophylline enhanced acute insulin secretion in vitro. Transplantation of up to 1000 ICCs into athymic and scid mice resulted in sparse growth of the epithelialike cells in the graft and only partial differentiation of the endocrine cells. Hyperglycaemia in diabetic recipients was not normalized. Thus, while functioning ICCs can be created from fetal sheep pancreas, they do not appear to be appropriate for transplantation to reverse diabetes in mice.     

兔胰岛细胞团颅内移植治疗糖尿病的研究

将新鲜制备的成年兔胰岛样细胞团经腰椎穿刺注入脊髓腔，采取小剂量多次颅内移植治疗糖尿病兔。术后不用免疫抑制剂，不用外源性胰岛素。结果１０只糖尿病兔在胰岛细胞团颅内移植后，３只显效，２只有效，无排斥反应及神经系统异常。     

Effects of ultraviolet B irradiation on fetal pig islet-like cell clusters

Abstract: Ultraviolet B (UV-B) irradiation of donor islets has previously been shown to result in the prolongation of their survival when transplanted into rodents. This study examined the in vitro and in vivo effects of UV-B irradiation on fetal pig islet-like cell clusters (ICCs), which like adult islets are being transplanted to reverse diabetes. Under control conditions, fetal pig ICCs were able to stimulate both human and pig peripheral blood mononuclear cells (PBMC) in mixed islet lymphocyte culture (MILC). Exposure of the ICCs to UV-B irradiation significantly reduced their ability to stimulate PBMC of both species in MILC when 600 J/m² but not lower doses (300 and 400 J/m²) of irradiation were applied. In contrast, all doses of UV-B irradiation were effective in inhibiting the ability of pig and human PBMC to stimulate human PBMC in a mixed lymphocytes culture (MLC). This demonstrates that UV-B irradiation is effective in reducing xeno immunogenicity of pig antigens. A toxic effect of all doses of UV-B irradiation on ICCs was demonstrated in vitro with a reduction in ³H-thymidine incorporation of 57, 71, 64, and 80% at 150, 300, 450, and 600 J/m², respectively. Toxicity of UV-B irradiation was also demonstrated when treated ICCs were transplanted beneath the renal capsule of SCID mice. The insulin content of the ICCs, 6 weeks after transplantation, was significantly reduced in the 600 J/m² group ( P <0.05). ICCs treated with UV-B irradiation (300 J/m²) in vitro and then transplanted beneath the renal capsule of BALB/c mice were rejected within 2 weeks as were untreated ICCs. Injection of cyclosporine (12.5 mg/kg/day) into these mice did not alter the results. It is concluded that UV-B irradiation is toxic to fetal pig ICCs and, in low dose, unable to prevent their rejection when transplanted into mice.     

Differentiation of CD133-positive pancreatic cells into insulin-producing islet-like cell clusters

Adult pancreatic stem and progenitor cells could represent an alternative source of insulin-producing tissue for diabetes treatment. In order to identify these cells, we have focused on the human pancreatic cells expressing cell surface molecule CD133, a marker of adult stem cells. We found that population of human CD133-positive pancreatic cells contains endocrine progenitors expressing neurogenin-3 and cells expressing human telomerase, ABCG2, Oct-3/4, Nanog, and Rex-1, markers of pluripotent stem cells. These cells were able to differentiate into insulin-producing cells in vitro and secreted C-peptide in a glucose-dependent manner. Based on our results, we suppose that the CD133 molecule represents another cell surface marker suitable for identification and isolation of pancreatic endocrine progenitors.     

Macrophage depletion improves survival of porcine neonatal pancreatic cell clusters contained in alginate macrocapsules transplanted into rats

Omer A, Keegan M, Czismadia E, De Vos P, Van Rooijen N, Bonner-Weir S and Weir GC. Macrophage depletion improves survival of porcine neonatal pancreatic cell clusters contained in alginate macrocapsules transplanted into rats. Xenotransplantation 2003; 10: 240–251. © Blackwell Munksgaard 2003
Background: Macrophages can accumulate on the surface of empty and islet-containing alginate capsules, leading to loss of functional tissue. In this study, the effect of peritoneal macrophage depletion on the biocompatibility of alginate macrocapsules and function of macroencapsulated porcine neonatal pancreatic cell clusters (NPCCs) was investigated. Methods: Clodronate liposomes were injected into the peritoneal cavities of normoglycemic Lewis rats 5 and 2 days before the transplantation. Empty or NPCC-containing Ca-alginate poly L -lysine (PLL)-coated macrocapsules were transplanted into the peritoneal cavities of rats injected with either clodronate liposomes or saline. On days 7, 14 and 21, samples were evaluated by immunohistochemistry for cellular immune responses on the surface of the macrocapsules and for macrophage populations in omental tissue. To assess the function of macroencapsulated NPCCs, insulin secretory responses to glucose and theophylline were measured after capsule retrieval. Results: In saline-injected control groups, all of the empty and NPCC-containing macrocapsules were overgrown with macrophages, this being especially severe on NPCC-containing macrocapsules. In the clodronate liposomes-injected group, the majority of the empty macrocapsules were free of macrophage accumulation and the NPCC-containing macrocapsules were less overgrown than in control animals. Higher insulin responses to glucose and theophylline were observed in NPCCs retrieved from rats injected with clodronate liposomes. Conclusion: We conclude that depletion of peritoneal macrophages with clodronate liposomes improve the survival of macroencapsulated NPCCs.     

A study of the xenoantigenicity of neonatal porcine islet-like cell clusters (NPCC) and the efficiency of adenovirus-mediated DAF (CD55) expression

BACKGROUND: The pig pancreas is considered to be the most suitable source of islets for xenotransplantation in patients with type I diabetes. The objective of this study was to assess the antigenicity of neonatal porcine islet-like cell clusters (NPCC), including the Galalpha1-3Galbeta1-4GlcNAc-R (alpha-Gal) and Hanganutziu-Deicher (H-D) antigens, and the pathway involved in human complement activation. The efficiency of expression of human decay-accelerating factor (DAF: CD55) on NPCC by adenoviral transduction was also examined, and the functional capacity of DAF was also estimated. METHODS: The deposition of human natural antibodies, immunoglobulin (Ig)G and IgM, and the expression of alpha-Gal and H-D antigens on NPCC were investigated by FACS analysis. The downregulation in the antigenicity to human natural antibodies, including the alpha-Gal and H-D antigens on NPCC by treatment with tunicamycin, PDMP and neuraminidase were also examined. In addition, complement-mediated islet lysis was examined using factor D-deficient and C1-deficient sera. An adenovirus encoding DAF under the control of the cytomegalovirus promoter, Ad.pCMV-DAF, was then constructed, and used for transducing NPCC. The amelioration of complement-dependent cytotoxicity of the NPCC by the transduced DAF was assessed as an in vitro hyperacute rejection model of a pig to human xenograft. RESULTS: The NPCC clearly expressed the alpha-Gal epitope, and the human natural antibodies, IgG and IgM, and the anti-H-D antibody also reacted with the NPCC. Treatment of NPCC with tunicamycin led to a drastic reduction in the extent of deposition of IgG, indicating the importance of N-linked sugars on the islets, presumably related to alpha-Gal expression on N-linked sugars. Neuraminidase treatment indicated the presence of, not only the H-D antigen, but also other sialic acid antigens which reacted with the human natural antibody, especially IgG. The complement deposition of factor B on NPCC was clear, and the alternative pathway-mediated NPCC killing accounted for approximately 30% of that by the total complement pathway. On the other hand, approximately 90% of the NPCC could be transduced to express DAF by the adenovector, Ad.pCMV-DAF. The expressed DAF showed an approximately 50-62% suppression in complement-dependent NPCC lysis. CONCLUSION: The origin of the antigenicity of NPCC is mainly N-linked sugars including alpha-Gal and sialic acid antigens, and NPCC expressed the transduced molecule in high efficiency by the adenovector.     

A novel human stem cell coculture system that maintains the survival and function of culture islet-like cell clusters

Islet-like cell clusters (ICCs) have been suggested to be a source of insulin-producing tissue for xenotransplantation in type 1 diabetes. We designed an approach to maintain the cultured rat pancreatic ICC survival and function, when cocultured with human umbilical cord mesenchymal stem cells (HUMSCs). HUMSCs in coculture have the ability to maintain ICC survival and function, for which number and insulin secretion of ICCs are increasing and lasting for 3 months, while ICCs gradually crash, which results in cell death after a period of 12 days of culture without HUMSCs. Cytokine protein array showed it has more than a twofold increase in levels of several cytokines (interleukin-6, tissue inhibitor of metalloproteinases-1, tissue inhibitor of metalloproteinases-2, monocyte chemoattractant protein-1, growth related oncogene, hepatocyte growth factor, insulin-like growth factor binding proteins 4, and interleukin-8) on coculture medium, implying an important role of these cytokines in this coculture system. These findings suggest that coculture with HUMSCs may have a significant potential to protect ICCs from damage during culture, and may be employed in a novel culture approach to maintain islet cell survival and function before transplantation.