Ziliang Ao

Maturation of Human Embryonic Stem Cell–Derived Pancreatic Progenitors Into Functional Islets Capable of Treating Pre-existing Diabetes in Mice

Diabetes is a chronic debilitating disease that results from insufficient production of insulin from pancreatic β-cells. Islet cell replacement can effectively treat diabetes but is currently severely limited by the reliance upon cadaveric donor tissue. We have developed a protocol to efficiently differentiate commercially available human embryonic stem cells (hESCs) in vitro into a highly enriched PDX1+ pancreatic progenitor cell population that further develops in vivo to mature pancreatic endocrine cells. Immature pancreatic precursor cells were transplanted into immunodeficient mice with streptozotocin-induced diabetes, and glycemia was initially controlled with exogenous insulin. As graft-derived insulin levels increased over time, diabetic mice were weaned from exogenous insulin and human C-peptide secretion was eventually regulated by meal and glucose challenges. Similar differentiation of pancreatic precursor cells was observed after transplant in immunodeficient rats. Throughout the in vivo maturation period hESC-derived endocrine cells exhibited gene and protein expression profiles that were remarkably similar to the developing human fetal pancreas. Our findings support the feasibility of using differentiated hESCs as an alternative to cadaveric islets for treating patients with diabetes.

Intraductal collagenase delivery into the human pancreas using syringe loading or controlled perfusion.

Effective intraductal delivery of the enzyme collagenase into the pancreas is crucial to the subsequent ability to isolate viable islets. Most clinical islet transplant centers load the enzyme into the pancreas by retrograde injection using a syringe following cannulation of the pancreatic duct. An alternative approach is to perfuse the pancreas via the pancreatic duct with collagenase solution using a recirculating perfusion device system. This provides control over perfusion pressures and collagenase temperature. This study reports on our evaluation of the delivery of Liberase™-HI into the pancreas of 14 consecutive adult multiorgan cadaveric donors. Alternate glands were procured and processed using an identical protocol with the exception of collagenase delivery. The first group of pancreases was loaded using the perfusion technique where cold (4°C) Liberase™-HI was perfused at 80 mmHg for 5 min after which the pressure was increased to 180 mmHg. The collagenase solution was then slowly warmed to 35°C, transferred to the dissociation chamber and mechanically dissociated, and then purified using discontinuous gradients of Ficoll. Pancreases in the second group were loaded with collagenase (28–32°C) using the syringe technique before mechanical dissociation and purification. There were no significant differences in pancreas cold ischemia, donor age, body mass index, maximum blood glucose, or serum amylase of the donors between the two groups. Mean collagenase digestion time in the digestion chamber was not different between the two groups; however, the amount of undigested tissue remaining after dissociation was significantly higher in the syringe-loaded group (15.3 ± 2.6 g vs. 4.6 ±2.1 g, mean ± SEM, p < 0.05). Postdigestion recovery of islets was 471 ± 83 × 103 IE in the perfusion group compared with 391 ± 57 × 103 IE for the syringe-loaded group. Postpurification recovery was higher in the perfused group (379 ± 45 vs. 251 ± 28 × 103 IE, p < 0.05, two-tailed paired t-test). No difference in in vitro islet viability was observed between the two groups following glucose perifusion with the calculated stimulation index of 4.6 ± 0.6 for the perfusion group and 4.2 ± 0.7 for the syringe-loaded group. Controlled perfusion via the pancreatic duct allows the effective delivery of the enzyme achieving maximal distension to all regions of the pancreas leading to an increased recovery of the islets with no detrimental effect on subsequent in vitro islet function.

Production of Functional Glucagon-Secreting α-Cells From Human Embryonic Stem Cells

OBJECTIVE Differentiation of human embryonic stem (hES) cells to fully developed cell types holds great therapeutic promise. Despite significant progress, the conversion of hES cells to stable, fully differentiated endocrine cells that exhibit physiologically regulated hormone secretion has not yet been achieved. Here we describe an efficient differentiation protocol for the in vitro conversion of hES cells to functional glucagon-producing α- cells. RESEARCH DESIGN AND METHODS Using a combination of small molecule screening and empirical testing, we developed a six-stage differentiation protocol for creating functional α-cells. An extensive in vitro and in vivo characterization of the differentiated cells was performed. RESULTS A high rate of synaptophysin expression (>75%) and robust expression of glucagon and the α-cell transcription factor ARX was achieved. After a transient polyhormonal state in which cells coexpress glucagon and insulin, maturation in vitro or in vivo resulted in depletion of insulin and other β-cell markers with concomitant enrichment of α-cell markers. After transplantation, these cells secreted fully processed, biologically active glucagon in response to physiologic stimuli including prolonged fasting and amino acid challenge. Moreover, glucagon release from transplanted cells was sufficient to reduce demand for pancreatic glucagon, resulting in a significant decrease in pancreatic α-cell mass. CONCLUSIONS These results indicate that fully differentiated pancreatic endocrine cells can be created via stepwise differentiation of hES cells. These cells may serve as a useful screening tool for the identification of compounds that modulate glucagon secretion as well as those that promote the transdifferentiation of α-cells to β-cells.

Reduced progression of diabetic microvascular complications with islet cell transplantation compared with intensive medical therapy.

Background. The effect of islet cell transplantation (ICT) on the progression of diabetic microvascular complications is not well understood. Methods. We have conducted a prospective, crossover, cohort study comparing ICT with intensive medical therapy on the progression of diabetic nephropathy, retinopathy, and neuropathy. Results. The rate of decline in glomerular filtration rate is slower after ICT than on medical therapy. There was significantly more progression of retinopathy in medically treated patients than post-ICT. There was a nonsignificant trend for improved nerve conduction velocity post-ICT. Conclusions. ICT is associated with less progression of microvascular complications than intensive medical therapy. Multicenter, randomized trials are needed to further study the role of ICT in slowing the progression of diabetic complications.

Different effects of FK506, rapamycin, and mycophenolate mofetil on glucose-stimulated insulin release and apoptosis in human islets.

Pancreatic islet transplantation has the potential to be an effective treatment for type 1 diabetes mellitus. While recent improvements have improved 1-year outcomes, follow-up studies show a persistent loss of graft function/survival over 5 years. One possible cause of islet transplant failure is the immunosuppressant regimen required to prevent alloimmune graft rejection. Although there is evidence from separate studies, mostly in rodents and cell lines, that FK506 (tacrolimus), rapamycin (sirolimus), and mycophenolate mofetil (MMF; CellCept) can damage pancreatic β-cells, there have been few side-by-side, multiparameter comparisons of the effects of these drugs on human islets. In the present study, we show that 24-h exposure to FK506 or MMF impairs glucose-stimulated insulin secretion in human islets. FK506 had acute and direct effects on insulin exocytosis, whereas MMF did not. FK506, but not MMF, impaired human islet graft function in diabetic NOD.scid mice. All of the immunosuppressants tested in vitro increased caspase-3 cleavage and caspase-3 activity, whereas MMF induced ER-stress to the greatest degree. Treating human islets with the GLP-1 agonist exenatide ameliorated the immunosuppressant-induced defects in glucose-stimulated insulin release. Together, our results demonstrate that immunosuppressants impair human β-cell function and survival, and that these defects can be circumvented to a certain extent with exenatide treatment.

Effect of exenatide on beta cell function after islet transplantation in type 1 diabetes.

Background. Islet transplantation can reduce or eliminate the need for insulin in patients with type 1 diabetes. Exenatide is a long acting analogue of Glucagon-like peptide-1 (GLP-1) that augments glucose induced insulin secretion, and may increase &bgr; cell mass. We evaluated the effect of exenatide on insulin secretion after islet transplantation. Methods. Eleven C-peptide positive islet cell recipients with elevated glucose levels were treated with exenatide for three months. Response was assessed by insulin requirements, meal tolerance tests, and hyperglycemic glucose clamps. Results. Ten patients responded to exenatide. Two patients who had not restarted insulin achieved good glycemic control and one patient who had received 5500 IE/kg in first islet infusion was able to stop insulin. Seven other patients decreased their insulin dose by 39% on exenatide. Hyperglycemic clamp studies showed a rise in second phase insulin release (before exenatide: 246±88 pM; during exenatide: 644±294 pM, P<0.01). Meal tolerance studies before and one month after stopping exenatide did not show a difference in glucose or C-peptide values. Nausea and vomiting were the major side effects. Conclusions. Exenatide stimulates insulin secretion in islet transplant recipients. It reduces insulin dose in some patients and may delay the need to resume insulin in others. We did not find any evidence of a trophic effect on islets.

A multi-year analysis of islet transplantation compared with intensive medical therapy on progression of complications in type 1 diabetes.

Background. We hypothesized that transplantation of islets into type 1 diabetics could improve outcomes of glucose metabolism, renal function, retinopathy, and neuropathy compared with intensive medical therapy. Methods. We conducted a prospective, crossover, cohort study of intensive medical therapy (group 1) versus islet cell transplantation (group 2) in 42 patients. All were enrolled in group 1 then 31 crossed over with group 2 when islet donation became available. Transplantation was performed by portal venous embolization of more than 12,000 islet equivalents/kg body weight under cover of immunosuppression with antithymocyte globulin, tacrolimus, and mycophenolate. Outcome measures were HbA1c, change in glomerular filtration rate (GFR), progression of retinopathy, and change in nerve conduction velocity. This report details interim analysis of outcomes after 34±18 months (group 1) and 38±18 months (group 2). Results. HbA1c (%) in group 1 was 7.5±0.9 versus 6.6±0.7 in group 2 (P<0.01). GFR (mL/min/month) declined in both groups (group 1 −0.45±0.7 vs. group 2 −0.12±0.7, P=0.1). Slope of the GFR decline in group 1 was significantly more than 0. Retinopathy progressed in 10 of 82 eyes in group 1 versus 0 of 51 in group 2 (P<0.01). Nerve conduction velocity (m/sec) remained stable in group 1 (47.8±5 to 47.1±5 m/sec) and group 2 (47.2±4.5 to 47.7±3.5). Conclusion. Islet transplantation yields improved HbA1c and less progression of retinopathy compared with intensive medical therapy during 3 years follow-up.

Glucose-Dependent Insulinotropic Polypeptide Is Expressed in Pancreatic Islet α-Cells and Promotes Insulin Secretion

BACKGROUND & AIMS Glucose-dependent insulinotropic polypeptide (GIP) and the proglucagon product glucagon-like peptide-1 (GLP-1) are gastrointestinal hormones that are released in response to nutrient intake and promote insulin secretion. Interestingly, a subset of enteroendocrine cells express both GIP and GLP-1. We sought to determine whether GIP also might be co-expressed with proglucagon in pancreatic alpha-cells. METHODS We assessed GIP expression via reverse-transcription polymerase chain reaction, in situ hybridization, and immunohistochemistry. We developed a novel bioassay to measure GIP release from isolated islets, compared the biological activities of full-length and truncated GIP, and assessed the impact of immunoneutralization of islet GIP on glucose-stimulated insulin secretion in isolated islets. RESULTS GIP messenger RNA was present in mouse islets; GIP protein localized to islet alpha-cells of mouse, human, and snake pancreas, based on immunohistochemical analyses. However, using a C-terminal GIP antibody, immunoreactivity was detected in islets from prohormone convertase (PC) 2 knockout but not wild-type mice. Bioactive GIP was secreted from mouse and human islets after arginine stimulation. In the perfused mouse pancreas, GIP(1-42) and amidated GIP(1-30) had equipotent insulinotropic actions. Finally, immunoneutralization of GIP secreted by isolated islets decreased glucose-stimulated insulin secretion. CONCLUSIONS GIP is expressed in and secreted from pancreatic islets; in alpha-cells, PC2 processes proGIP to yield a truncated but bioactive form of GIP that differs from the PC1/3-derived form from K-cells. Islet-derived GIP promotes islet glucose competence and also could support islet development and/or survival.

Suppression of p38 MAPK and JNK via Akt-mediated inhibition of Apoptosis Signal regulating Kinase 1 constitutes a core component of the β-cell pro-survival effects of glucose-dependent insulinotropic polypeptide

Glucose-dependent insulinotropic polypeptide (GIP) potentiates glucose-stimulated insulin secretion, insulin biosynthesis, and β-cell proliferation and survival. In previous studies GIP was shown to promote β-cell survival by modulating the activity of multiple signaling modules and regulating gene transcription of pro- and anti-apoptotic bcl-2 family proteins. We have now evaluated the mechanisms by which GIP regulates the dynamic interactions between cytoplasmic bcl-2 family members and the mitochondria in INS-1 cells during apoptosis induced by treatment with staurosporine (STS), an activator of the mitochondria-mediated apoptotic pathway. STS induced translocation of bad and bimEL, activation of mitochondrial bax, release of mitochondrial cytochrome c, cleavage of caspase-3, and apoptosis. Each response was significantly diminished by GIP. Using selective enzyme inhibitors, overexpression of dominant-negative Akt, and Akt siRNA, it was demonstrated that GIP promoted β-cell survival via Akt-dependent suppression of p38 MAPK and JNK and that combined inhibition was sufficient to explain the entire pro-survival responses to GIP during STS treatment. This signaling pathway also explained the pro-survival effects of GIP on INS-1 cells exposed to two other promoters of stress: thapsigargin (endoplasmic reticulum stress) and etoposide (genotoxic stress). Importantly, we discovered that GIP suppressed p38 MAPK and JNK via Akt-mediated changes in the phosphorylation state of the apoptosis signal-regulating kinase 1 in INS-1 cells and human islets, resulting in inhibition of its activity. Inhibition of apoptosis by GIP is therefore mediated via a key pathway involving Akt-dependent inhibition of apoptosis signal-regulating kinase 1, which subsequently prevents the pro-apoptotic actions of p38 MAPK and JNK.

The effect of medical therapy and islet cell transplantation on diabetic nephropathy : An interim report

Background. The effect of islet cell transplantation (ICT) on renal function in type 1 diabetes is uncertain and some recent studies report a significant decline in estimated glomerular filtration rate (GFR) and worsening of albuminuria. Methods. We are conducting a prospective crossover study comparing medical treatment with islet transplantation on the progression of diabetic complications, including renal function. The primary endpoint is change in GFR measured by 99mTc-diethylenetriaminepentaacetate with secondary endpoints including estimated GFR and albumin excretion. Results. We have followed 21 patients after islet transplantation a median of 29 months (range 13–45) and compared their results with medically treated patients followed a median 29.5 months (range 13–56). There is no difference in the rate of decline in measured GFR between medically treated patients (–0.35±0.89; 95% CI: –0.57 to –0.13 mL/min/month/1.73 m2) and those after ICT (–0.31±1.18; 95% CI: –0.61 to –0.01) and neither is significantly different from that expected for the general population. The rate of decline in our estimated GFR results is lower than that reported in other studies and we did not find any worsening of albuminuria. Conclusions. We do not find evidence of worsening of renal function after islet transplantation compared with medically treated patients.