Lindy Williams

Safety and Viability of Microencapsulated Human Islets Transplanted Into Diabetic Humans

OBJECTIVE Transplantation of insulin-producing cells placed inside microcapsules is being trialled to overcome the need for immunosuppressive therapy. RESEARCH DESIGN AND METHODS Four type 1 diabetic patients with no detectable C-peptide received an intraperitoneal infusion of islets inside microcapsules of barium alginate (mean 178,200 islet equivalents on each of eight occasions). RESULTS C-peptide was detected on day 1 post-transplantation, and blood glucose levels and insulin requirements decreased. C-peptide was undetectable by 1–4 weeks. In a multi-islet recipient, C-peptide was detected at 6 weeks after the third infusion and remains detectable at 2.5 years. Neither insulin requirements nor glycemic control was affected. Capsules recovered at 16 months were surrounded by fibrous tissue and contained necrotic islets. No major side effects or infection occurred. CONCLUSIONS While allografting of encapsulated human islets is safe, efficacy of the cells needs to improve for the therapy to make an impact on the clinical scene.

Differentiation of transplanted microencapsulated fetal pancreatic cells.

Background. Fetal &bgr; cells are a potential form of cell therapy for type 1 diabetes. To protect transplanted cells from cellular immune attack, microencapsulation using barium alginate can be employed. Whether microencapsulated fetal pancreatic cells will differentiate as occurs with nonencapsulated fetal pancreatic cells is presently unknown. It is suggested that such differentiation would occur in encapsulated cells, similar to previous experiments conducted using encapsulated embryonic stem cells. Methods. Streptozotocin-induced diabetic severe combined immunodeficient mice were transplanted with 5,000 to 38,000 fetal pig islet-like cell clusters (ICCs) within barium alginate microcapsules of diameter 300, 600, or 1000 &mgr;m. Viability, insulin secretion, and content of encapsulated cells were measured prior to transplantation. Blood glucose levels (BGL) were measured twice weekly and porcine C-peptide monthly. Encapsulated cells were recovered from mice at 6 months posttransplantation for analysis. Results. Encapsulated cells became glucose responsive and normalized BGL within 13 to 68 days posttransplantation, with 5,000 to 10,000 ICCs required. Microcapsule diameter did not affect the time required to achieve normoglycemia. BGL remained normal for the 6-month duration of the experiments. After removal of grafts at 25 weeks posttransplantation, glucose stimulated insulin secretion of the explants was enhanced 96-fold, insulin content was enhanced 34-fold, and the percentage of insulin and glucagon positive cells increased 10-fold and threefold, respectively, from the time of transplantation. Conclusions. This study demonstrates that fetal pancreatic cells differentiate and function normally when placed within barium alginate microcapsules and transplanted.

Reduced Immunogenicity of First-Trimester Human Fetal Pancreas

OBJECTIVE—The use of human fetal pancreatic tissue may provide a potential source of transplantable β-cells as a therapy for type 1 diabetes. Human fetal pancreas has a remarkable capacity to grow and differentiate in vivo and has been shown to reverse diabetes in rodents. However, it is known that human fetal pancreas obtained from the second trimester of gestation is immunogenic and is rejected after transplantation. Tissue obtained from earlier stages might prove to be immune privileged, as has been shown for other tissues. RESEARCH DESIGN AND METHODS—In this study, we determined the immunogenicity of human fetal pancreatic tissue obtained from the first trimester of gestation in a humanized mouse model. A microarray study of immunoregulatory gene expression in first- and second-trimester human fetal pancreas was also undertaken. RESULTS—The analysis of transplanted human fetal pancreata revealed a significantly decreased immunogenicity of the first-trimester tissue. The first-trimester grafts showed only limited cellular infiltration and contained numerous insulin-positive cells, whereas second-trimester tissue was completely infiltrated and rejected. Furthermore an analysis of immunoregulatory genes expressed in first- and second-trimester human fetal pancreas by microarray demonstrated the upregulation of several key immunoregulatory genes in the second-trimester tissue. This might account for the reduced immunogenicity of the younger tissue. CONCLUSIONS—Our results provide the first indication that the use of first-trimester human fetal pancreas for transplantation might increase the survival of the grafts and might decrease the requirement for immunosuppressive drugs.

Comparison of size, viability, and function of fetal pig islet-like cell clusters after digestion using collagenase or liberase.

Liberase is a highly purified blend of collagenases that has been specifically developed to eliminate the numerous problems associated with the conventional use of crude collagenase when isolating islet-like cell clusters (ICCs) from pancreases of different species. The influence of Liberase on yield, size, viability, and function of ICCs has been documented when this enzyme was used to digest adult but not fetal pancreases. In this study, we compared the effects of collagenase and Liberase on fetal pig ICCs. A total of eight fetal pig pancreas digestions were analyzed. Fetuses were obtained from Large White Landrace pigs of gestational age 80 ± 2.1 days. The pancreases were digested with either 3 mg/ml collagenase P or 1.2 mg/ml Liberase HI. The time taken to digest the pancreas was shorter for collagenase when compared with Liberase (22 ± 2 vs. 31 ± 2 min). The size of ICCs was similar for both collagenase (83 ± 0.5 μm) and Liberase (79 ± 0.4 μm) as was the number of ICCs produced per pancreas (7653 ± 1297 vs. 8101 ± 1177). Viability, as assessed using fluorescent markers, was slightly greater for Liberase (79 ± 1% vs. 76 ± 1%, p < 0.05). Responsiveness to β-cell stimulus (20 mM KCl) was similar for both methods of isolation, as was the insulin content of the ICCs, both in vitro and at 1 month after transplantation of 1500 ICCs beneath the renal capsule of immunoincompetent mice. Despite the high content of endotoxins in collagenase, the above results show that this enzyme was equally as efficient as Liberase in isolating functional ICCs from fetal pig pancreas.

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, β-cells constitute only 6–8% of the cells, in contrast to 65% in an adult pig islet. Attempts to purify fetal β-cells from cell clusters and compare their function to that of ICCs have not been shown previously. β-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 β-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 β-cells differentiate. This finding may have implications for stem cell therapy, as other cell types, other than purified β-cells, may be necessary for appropriate function in vivo.

Porcine pancreatic icosapeptide as a marker of graft survival and rejection in xenotransplantation.

Abstract:  The previous study showed that it is possible to monitor the viability of xenografted fetal pig pancreas in the first 3 weeks after transplantation in a normoglycemic immunoincompetent mouse [ 1 ]. This is achieved by measuring serum levels of porcine pancreatic icosapeptide (PI) in the host using a specific immunoassay. PI is secreted from the pancreatic polypeptide (PP) cell, the first mature endocrine cell of the fetal pig pancreas. Insufficient insulin is produced by the graft at this time to use it as a marker of graft viability. We have now examined the usefulness of PI to monitor graft survival in diabetic immunoincompetent mice and in immunosuppressed mice. Fetal porcine pancreatic tissue was transplanted beneath the renal capsule of streptozotocin‐diabetic non‐obese diabetic severe combined immunodeficient mice (NOD‐SCID) and BALB/c mice given cyclosporin (CsA) for 21 or 10 days, respectively. Blood and grafts were analyzed periodically over 15 and 3 weeks, respectively, for porcine PI and C‐peptide/insulin. In the diabetic mice, porcine PI was detected in sera up to 2 weeks following transplantation and was absent thereafter. Serum porcine C‐peptide was detectable at 3 weeks and reached maximum levels at 12 to 15 weeks. PI content of the grafts was highest at day 4 and 3 weeks and lowest from 9 weeks onwards while insulin levels in the graft were highest from 9 weeks onwards. In BALB/c mice immunosuppressed with CsA, serum PI was detectable for 21 days. In the mice given CsA only for 10 days, serum PI was detectable for that time, but not on day 14 although PI was detectable in the graft. In mice given no CsA, PI was undetectable in serum at any time although PI was present in the graft at day 4. The presence of porcine PI in sera is a marker of graft survival of fetal porcine pancreatic tissue in diabetic immunocompetent mice in the first 2 weeks after transplantation. Its absence in immunosuppressed mice in this period is an early indicator of graft rejection.

Outcomes for Islet Transplantation in Donation After Circulatory Death compared with Donation after Brain Death in Australia

Introduction Islet cell transplantation has become a clinically accepted transplant technique providing long-term insulin independence, restoring normoglycaemia and treating T1D patient’s severe hypoglycaemic unawareness. Unfortunately, the significant lack of organ donors results in patients remaining on the waitlist for years. Donation after Circulatory Death (DCD) donors have been reported by other groups as having up to 60% of selected DCD islet preparations proceed to transplant with similar outcomes to donation after brain death (DBD) organs. We review our isolation and transplantation outcomes from DCD donors and compare these to DBD donors over the same time period in the Australian program. Materials and Methods Islet donor pancreata were compared from the Australian National Islet Transplant program with direct comparison of organ donor, islet isolation variables and transplantation outcomes. Donor variables included; sex, age, weight, BMI, cause of death, CIT, WIT, pancreas weight. Isolation outcomes included; pre- and post-purification islet yield, post-culture yield, purity and viability, and transplantation outcomes such as islet number transplanted and abrogation of severe hypoglycaemic unawareness. Results and Discussion A total of 27 DCD and 73 DBD islet donor pancreata were compared with no significant differences seen in donor characteristics between DCD and DBD. However, upon evaluation of the isolation outcomes it showed that post-purification yield (IEQ) was significantly lower from the DCD group (146,518±28,971) compared to the DBD group (256,986±17,652; P=0.001). Post-purification yield per gram of pancreas was also lower from the DCD group (2,154±504 vs. 2,681±372 IEQ/g); and was significant (P<0.0001). Post-culture yields in terms of total IEQ and IEQ per gram of pancreas were more than four times lower from DCD pancreata (37,634±27,786 IEQ and 455±305 IEQ/g) compared to DBD pancreata (234,860±18,132 IEQ and 2,280±199 IEQ/g), which reached extreme significance (P<0.0001). The quality and functionality of DCD and DBD islets were also significantly different in terms of the viability (%) – P=0.017 (higher in DBD than DCD), purity (%) – P=0.001 (higher in DBD than DCD), stimulation index, and beta cell viability index outcomes were not different. The proportion of DCD islets transplanted (1/27) was significantly lower than DBD (29/73) going to transplant (OR, 0.1093; 95% CI; P=0.001). Conclusion In the Australian setting with vast distances to ship pancreata we have had poorer outcomes from DCD pancreata for islet isolation and have thus far not yielded outcomes comparable to those from our DBD donors. Earlier intervention, the use of ante mortem heparin and faster logistics in transport may not only improve the DCD organs for transplantation but also help alleviate donor shortages allowing treatment of those with T1DM and severe hypoglycaemic unawareness.

Standardized Whole-Blood Immunophenotyping Panels on Flow Cytometry for Transplant Recipients and Clinical Trials

Introduction Immunophenotyping of whole blood by flow cytometry is a reliable, fast, and easy method used to obtain a large amount of information on the effects and outcomes of different treatments in transplantation on immune phenotype with minimal impact on the patient. Aim: 1) To establish whole-blood immunophenotyping panels for transplant recipients, 2) To develop a concise method involving the standardisation of reagents, sample handling, instrument setup and data analysis. Methods Absolute cell count (TruCount) and seven leukocyte-profiling panels containing 8-10 marker-antigens (46 of which were unique within the panels) consisting of subsets and/or status of granulocytes, monocytes, DCs, B, NK,, and T cells including Tregs and NKT were used to monitor the immune profiles of paediatric kidney transplant and adult islet cell transplant recipients. Whole-blood samples were stained and acquired on a BD-LSRFortessa and Flowjo was used for data analysis. BD™ Cytometer Setup and Tracking beads monitored cytometer performance. Sample staining was performed within 2 hours of blood sample collection. The accuracy and variability of these panels were determined and 100-300 &mgr;l whole-blood was used for each panel. Results: The 46 antibodies were titrated and the staining index (SI) was calculated (CD45BUV395 in Fig.1A) for panel optimisation. Application settings on a BD LSRFortessa, measurement of the spillover spreading matrix (SSM) for each panel [SSM in Panel 3 (Tab.1)], and optimal antibody quantity for the panel-cocktail were established. Auto-analysis templates and gating strategies (Panel 1 in Fig.1B) to target subsets of immune-cells were set up and blood sample collection, preparation, antibody cocktails, and staining protocols were standardised. 9 paediatric kidney transplant patients, 4 islet transplant patients (which are to be followed until two years post third islet transplant), 13 T1D patients and 8 control samples have been evaluated to date. The ability to identify consistent immune subsets across all panels over 6 months (Fig.1C from TruCount Panel) was achieved, and was used to longitudinally track the proportions of cell populations in transplant patients (Fig.1D Panel 1). Conclusion We standardised immune panels and procedures for absolute cell numbers and multiple subsets of immune cells for monitoring a range of individuals including healthy controls, paediatric kidney recipients, T1D, and islet transplant recipients. We have demonstrated that immunophenotyping by flow cytometry is a reliable, consistent, and fast technique that allows the detection of changes in absolute cell numbers of leukocyte subsets from any recipients’ whole blood. This immunophenotyping by flow cytometry is a non-invasive technique which requires less than 1.5ml of whole blood and may be a useful tool for monitoring changes in the immune profile of individuals in a range of clinical trials. Birgit Sawitzki. Mathias Streitz. Figure. No caption available. Table. No title available.