Background. Bone marrow-derived mesenchymal stem cells (MSCs) are known to produce vascular endothelial growth factor. We hypothesize that co-transplantation of MSCs and islets promotes revascularization and improves islet graft function. Methods. Lewis rat islets were infused into the liver of streptozotocin-diabetic syngeneic recipients or transplanted under the renal capsule of nonobese diabetic severe combined immunodeficiency (NOD SCID) mice with MSCs isolated from Lewis bone marrow and expanded in culture. Results. Co-transplantation of 500 islets and 10⁷ MSCs (islet-MSCs) reversed diabetes in all eight recipients, whereas islet-alone transplantation achieved euglycemia in 3 of 10 recipients. With 300 islets, five of nine islet-MSCs and 1 of 10 islets-alone recipients reversed diabetes. Results of intravenous glucose tolerance tests performed on day 56 were significantly better in islet-MSCs than islet-alone recipients. One week after transplantation, well-preserved islet structure and higher number of capillaries were found in the liver of islet-MSCs recipients, whereas islet-alone grafts were fragmented with very few capillaries. Islets showed a similar morphology when transplanted with MSCs in nonobese diabetic severe combined immunodeficiency mice with a significantly higher capillary per β-cell ratio than that in islet-alone grafts (0.135±0.046 vs. 0.052±0.028 capillary segments per β-cell, P<0.01). One week after transplantation, islets were surrounded by MSCs labeled with carboxyfluorescein succinimidyl ester or Qdot nanocrystals, and some labeled MSCs positively stained for vascular endothelial growth factor or von Willebrand factor. Conclusion. Our results demonstrate the improvement of islet graft morphology and function by co-transplantation with MSCs. This improvement is attributable, at least in part, to the promotion of graft revascularization mediated by MSCs.

Induction of hematopoietic chimerism and subsequent donor‐specific immune tolerance via bone marrow transplantation is an ideal approach for islet transplantation to treat type‐1 diabetes. We examined the potential of mesenchymal stem cells (MSCs) in the induction of chimerism and islet allograft tolerance without the incidence of graft‐versus‐host disease (GVHD). Streptozotocin‐diabetic rats received a conditioning regimen consisting of antilymphocyte serum and 5 Gy total body irradiation, followed by an intraportal co‐infusion of allogeneic MSCs, bone marrow cells (BMCs) and islets. Although all the recipients rejected the islets initially, half of them developed stable mixed chimerism and donor‐specific immune tolerance, shown by the engraftment of donor skin and second‐set islet transplants and acute rejection of a third‐party skin. The engraftment of the primary islet allografts with stable chimerism was achieved by the addition of a 2‐week peritransplant administration of 15‐deoxyspergualin (DSG). Without MSCs, none of the recipients treated with DSG developed chimerism or reversal of diabetes. GVHD was not observed in any of the recipients infused with MSCs (0/15), whereas it occurred in 4/11 recipients without MSCs. These results indicate a potential use of MSCs for induction of hematopoietic chimerism and subsequent immune tolerance in clinical islet transplantation.

Background. The activation of p38 mitogen-activated protein kinases (MAPK) is implicated in cold ischemia-reperfusion injury of donor organs. The islet isolation process, from pancreas procurement through islet collection, may activate p38MAPK leading to cytokine release and islet damage. This damage may be prevented by treating pancreata with a p38MAPK inhibitor (p38IH) before cold preservation. Methods. Pancreata removed from Beagle dogs were infused with University of Wisconsin solution containing the p38IH, SB203580, and Pefabloc (n=6) or vehicle (dimethyl sulfoxide and Pefabloc) alone (n=7), through the pancreatic duct and preserved using the two-layer method. After 20 to 22 hr, islets were isolated and 3000 IEQ/kg were autotransplanted into the corresponding dog to monitor glucose metabolism. Results. p38IH-treated pancreata yielded significantly more islets than control pancreata (IEQ/g: 2134±297 vs. 1477±145 IEQ/g or 65,012±9385 vs. 45,700±5103 IEQ/pancreas; P<0.05). Apoptotic β-cell percentages assessed by laser scanning cytometry were lower in p38IH-treated than the controls (44%±9.4% vs. 61.6%±4.8%, P<0.05). Tumor necrosis factor-α expression assessed by real-time reverse transcription polymerase chain reaction was significantly lower in the p38IH-treated group than controls. All dogs (3000 IEQ/kg) transplanted with p38IH-treated islets (n=5) became euglycemic versus four of five dogs that received untreated islets. Plasma C-peptide levels after glucagon challenge were higher in animals receiving p38IH-treated islets (n=5) versus untreated islets (n=4) (0.40±0.78 vs. 0.21±0.05 ng/mL, P<0.05). Conclusions. Infusion of pancreata with University of Wisconsin solution containing p38IH through the duct before preservation suppresses cytokine release, prevents β-cell apoptosis, and improves islet yield significantly with no adverse effect on islet function after transplantation. p38IH treatment of human pancreata may improve islet yield for use in clinical transplantation.

Assays for assessing human islet cell quality, which provide results before transplantation, would be beneficial to improve the outcomes for islet transplantation therapy. Parameters such as percent β-cell apoptosis and cell composition are found to vary markedly between different islet preparations and may serve as markers of islet quality. We have developed fluorescence-based assays using laser scanning cytometry for assessing β-cell apoptosis and islet cell composition on serial sections of intact isolated islets. Isolated human islets were fixed in formalin and embedded in paraffin. Serial sections were immunostained for the pancreatic hormones and acinar and ductal cell markers. DNA fragmentation was used to label apoptotic cells. Stained cells were quantified using an iCys laser scanning cytometer. Islet preparations from 102 human pancreatic islet isolations were analyzed. For the whole set of islet preparations, we found a mean islet cell composition of 54.5%±1.2% insulin-positive, 33.9%±1.2% glucagon, 12.1%±0.7% somatostatin, and 1.5%±0.2% pancreatic polypeptide-positive cells. The apoptotic β cells were 2.85%±0.4% with a range of 0.27% to 18.3%. The percentage of apoptotic β cells correlated well (P<0.0001, n=59) with results obtained in vivo by transplantation of the corresponding islets in diabetic NODscid mice. The analysis of whole, nondissociated islets for cell composition and β-cell apoptosis using laser scanning cytometry gives reliable and reproducible results and could be performed both before islet transplantation and on preserved cell blocks at any time in future. Thus, they can be a powerful tool for islet quality assessment.