The enzyme α1,3-galactosyltransferase (α1,3GT or GCTA1) synthesizes α1,3-galactose (α1,3Gal) epitopes (Galα1,3Galβ1,4GlcNAc-R), which are the major xenoantigens causing hyperacute rejection in pig-to-human xenotransplantation. Complete removal of α1,3Gal from pig organs is the critical step toward the success of xenotransplantation. We reported earlier the targeted disruption of one allele of the α1,3GT gene in cloned pigs. A selection procedure based on a bacterial toxin was used to select for cells in which the second allele of the gene was knocked out. Sequencing analysis demonstrated that knockout of the second allele of the α1,3GT gene was caused by a T-to-G single point mutation at the second base of exon 9, which resulted in inactivation of the α1,3GT protein. Four healthy α1,3GT double-knockout female piglets were produced by three consecutive rounds of cloning. The piglets carrying a point mutation in the α1,3GT gene hold significant value, as they would allow production of α1,3Gal-deficient pigs free of antibiotic-resistance genes and thus have the potential to make a safer product for human use.The enzyme α1,3-galactosyltransferase (α1,3GT or GGTA1) synthesizes α1,3Gal epitopes (Galα1,3Galβ1,4GlcNAc-R) on the cell surface of almost all mammals with the exception of humans, apes, and Old World monkeys (1). α1,3Gal epitopes are the major xenoantigens causing hyperacute rejection (HAR) in pig-to-human xenotransplantation (2-4). Many reports have also indicated that α1,3Gal epitopes are involved in acute vascular rejection (AVR) of xenografts (4-6). Piglets with α1,3GT heterozygous knockout have been cloned Copyright © 2003 by our group (7) and another team (8) in the last year. To produce homozygous α1,3GT knockout piglets by natural breeding, assuming both male and female heterozygous knockout pigs are available at the same time and are fertile, is feasible but takes up to 12 months. However, by using a second-round knockout and cloning strategy, we could save up to 6 months and all cloned piglets would be α1,3GT double knockout (DKO). We have selected and enriched for α1,3GT DKO cells by using a bacterial toxin, toxin A from Clostridium difficile, which binds with high affinity to α1,3Gal epitopes and produces a cytotoxic effect on cells that are α1,3Gal-positive (9). Toxin A uses α1,3Gal epitopes as a cell surface receptor and causes "rounding" and lifting of the α1,3Gal-positive cells from the surface of the growth vessel (10, 11).Heterozygous α1,3GT knockout fetal fibroblasts, 657A-I11 1-6 cells, were isolated from a day-32 pregnancy as described in (7). To avoid using a second antibiotic-resistance gene as a selection marker, we constructed an ATG (start codon)-targeting α1,3GT knockout vector, pPL680 (12), which also contains a neo gene, to knock out the second allele of the α1,3GT gene. 657A-I11 1-6 cells were transfected by electroporation with pPL680 and selected for the α1,3Gal-negative phenotype with purified C. difficile toxin A (13). One colony (680B1) was isolated and expanded af...

Approximately 285 million people worldwide suffer from diabetes, with insulin supplementation as the most common treatment measure. Regenerative medicine approaches such as a bioengineered pancreas has been proposed as potential therapeutic alternatives. A bioengineered pancreas will benefit from the development of a bioscaffold that supports and enhances cellular function and tissue development. Perfusion-decellularized organs are a likely candidate for use in such scaffolds since they mimic compositional, architectural and biomechanical nature of a native organ. In this study, we investigate perfusion-decellularization of whole pancreas and the feasibility to recellularize the whole pancreas scaffold with pancreatic cell types. Our result demonstrates that perfusion-decellularization of whole pancreas effectively removes cellular and nuclear material while retaining intricate three-dimensional microarchitecture with perfusable vasculature and ductal network and crucial extracellular matrix (ECM) components. To mimic pancreatic cell composition, we recellularized the whole pancreas scaffold with acinar and beta cell lines and cultured up to 5 days. Our result shows successful cellular engraftment within the decellularized pancreas, and the resulting graft gave rise to strong up-regulation of insulin gene expression. These findings support biological utility of whole pancreas ECM as a biomaterials scaffold for supporting and enhancing pancreatic cell functionality and represent a step toward bioengineered pancreas using regenerative medicine approaches.

Diabetes is a disease of epidemic proportions and is

Islet transplantation is a promising treatment for diabetes but longterm success is limited by progressive graft loss. Aggregates of the beta cell peptide islet amyloid polypeptide (IAPP) promote beta cell apoptosis and rapid amyloid formation occurs in transplanted islets. Porcine islets are an attractive alternative islet source as they demonstrate long-term graft survival. We compared the capacity of transplanted human and porcine islets to form amyloid as an explanation for differences in graft survival. Human islets were transplanted into streptozotocin-diabetic immune-deficient mice. Amyloid deposition was detectable at 4 weeks posttransplantation and was associated with islet graft failure. More extensive amyloid deposition was observed after 8 weeks. By contrast, no amyloid was detected in transplanted neonatal or adult porcine islets that had maintained normoglycemia for up to 195 days. To determine whether differences in IAPP sequence between humans and pigs could explain differences in amyloid formation and transplant viability, we sequenced porcine IAPP. Porcine IAPP differs from the human sequence at 10 positions and includes substitutions predicted to reduce its amyloidogenicity. Synthetic porcine IAPP was considerably less amyloidogenic than human IAPP as determined by transmission electron microscopy, circular dichroism, and thioflavin T binding. Viability assays indicated that porcine IAPP is significantly less toxic to INS-1 beta cells than human IAPP. Our findings demonstrate that species differences in IAPP sequence can explain the lack of amyloid formation and improved survival of transplanted porcine islets. These data highlight the potential of porcine islet transplantation as a therapeutic approach for human diabetes.