Chengyang Liu

Epigenomic plasticity enables human pancreatic α to β cell reprogramming

Insulin-secreting β cells and glucagon-secreting α cells maintain physiological blood glucose levels, and their malfunction drives diabetes development. Using ChIP sequencing and RNA sequencing analysis, we determined the epigenetic and transcriptional landscape of human pancreatic α, β, and exocrine cells. We found that, compared with exocrine and β cells, differentiated α cells exhibited many more genes bivalently marked by the activating H3K4me3 and repressing H3K27me3 histone modifications. This was particularly true for β cell signature genes involved in transcriptional regulation. Remarkably, thousands of these genes were in a monovalent state in β cells, carrying only the activating or repressing mark. Our epigenomic findings suggested that α to β cell reprogramming could be promoted by manipulating the histone methylation signature of human pancreatic islets. Indeed, we show that treatment of cultured pancreatic islets with a histone methyltransferase inhibitor leads to colocalization of both glucagon and insulin and glucagon and insulin promoter factor 1 (PDX1) in human islets and colocalization of both glucagon and insulin in mouse islets. Thus, mammalian pancreatic islet cells display cell-type-specific epigenomic plasticity, suggesting that epigenomic manipulation could provide a path to cell reprogramming and novel cell replacement-based therapies for diabetes.

Epigenetic regulation of the DLK1-MEG3 microRNA cluster in human type 2 diabetic islets.

Type 2 diabetes mellitus (T2DM) is a complex disease characterized by the inability of the insulin-producing β cells in the endocrine pancreas to overcome insulin resistance in peripheral tissues. To determine if microRNAs are involved in the pathogenesis of human T2DM, we sequenced the small RNAs of human islets from diabetic and nondiabetic organ donors. We identified a cluster of microRNAs in an imprinted locus on human chromosome 14q32 that is highly and specifically expressed in human β cells and dramatically downregulated in islets from T2DM organ donors. The downregulation of this locus strongly correlates with hypermethylation of its promoter. Using HITS-CLIP for the essential RISC-component Argonaute, we identified disease-relevant targets of the chromosome 14q32 microRNAs, such as IAPP and TP53INP1, that cause increased β cell apoptosis upon overexpression in human islets. Our results support a role for microRNAs and their epigenetic control by DNA methylation in the pathogenesis of T2DM.

B lymphocyte–directed immunotherapy promotes long-term islet allograft survival in nonhuman primates

We found that an induction immunotherapy regimen consisting of rabbit anti-thymocyte globulin (Thymoglobulin) and the monoclonal antibody to CD20 rituximab (Rituxan) promoted long-term islet allograft survival in cynomolgus macaques maintained on rapamycin monotherapy. B lymphocyte reconstitution after rituximab-mediated depletion was characterized by a preponderance of immature and transitional cells, whose persistence was associated with long-term islet allograft survival. Development of donor-specific alloantibodies was abrogated only in the setting of continued rapamycin monotherapy.

MicroRNA-7 Regulates the mTOR Pathway and Proliferation in Adult Pancreatic β-Cells

Elucidating the mechanism underlying the poor proliferative capacity of adult pancreatic β-cells is critical to regenerative therapeutic approaches for diabetes. Here, we show that the microRNA (miR)-7/7ab family member miR-7a is enriched in mouse adult pancreatic islets compared with miR-7b. Remarkably, miR-7a targets five components of the mTOR signaling pathway. Further, inhibition of miR-7a activates mTOR signaling and promotes adult β-cell replication in mouse primary islets, which can be reversed by the treatment with a well-known mTOR inhibitor, rapamycin. These data suggest that miR-7 acts as a brake on adult β-cell proliferation. Most importantly, this miR-7–mTOR proliferation axis is conserved in primary human β-cells, implicating miR-7 as a therapeutic target for diabetes.

The use of non-heart-beating donors for isolated pancreatic islet transplantation

Recent improvements in isolated islet transplantation indicate that this therapy may ultimately prove applicable to patients with type I diabetes. An obstacle preventing widespread application of islet transplantation is an insufficient supply of cadaveric pancreata. Non-heart-beating donors (NHBDs) are generally not deemed suitable for whole-organ pancreas donation and could provide a significant source of pancreata for islet transplantation. Isolated pancreatic islets prepared from 10 NHBDs were compared with those procured from 10 brain-dead donors (BDDs). The success of the isolation for the two groups was analyzed for preparation purity, quality, and recovered islet mass. The function of NHBD and BDD islets was evaluated using in vitro and in vivo assays. On the basis of the results of this analysis, an NHBD isolated islet allograft was performed in a type I diabetic. The recovery of islets from NHBDs was comparable to that of control BDDs. In vitro assessment of NHBD islet function revealed function-equivalent BDD islets, and NHBD islets transplanted to non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice efficiently reversed diabetes. Transplantation of 446,320 islet equivalents (IEq) (8,500 IEq/kg of recipient body weight) from a single NHBD successfully reversed the diabetes of a type I diabetic recipient. Normally functioning pancreatic islets can be isolated successfully from NHBDs. A single donor transplant from an NHBD resulted in a state of stable insulin independence in a type I diabetic recipient. These results indicate that NHBDs may provide an as yet untapped source of pancreatic tissue for preparation of isolated islets for clinical transplantation.

Transplantation for Type I Diabetes: Comparison of Vascularized Whole-Organ Pancreas With Isolated Pancreatic Islets

Objective:We sought to compare the efficacy, risks, and costs of whole-organ pancreas transplantation (WOP) with the costs of isolated islet transplantation (IIT) in the treatment of patients with type I diabetes mellitus. Summary Background Data:A striking improvement has taken place in the results of IIT with regard to attaining normoglycemia and insulin independence of type I diabetic recipients. Theoretically, this minimally invasive therapy should replace WOP because its risks and expense should be less. To date, however, no systematic comparisons of these 2 options have been reported. Methods:We conducted a retrospective analysis of a consecutive series of WOP and IIT performed at the University of Pennsylvania between September 2001 and February 2004. We compared a variety of parameters, including patient and graft survival, degree and duration of glucose homeostasis, procedural and immunosuppressive complications, and resources utilization. Results:Both WOP and IIT proved highly successful at establishing insulin independence in type I diabetic patients. Whole-organ pancreas recipients experienced longer lengths of stay, more readmissions, and more complications, but they exhibited a more durable state of normoglycemia with greater insulin reserves. Achieving insulin independence by IIT proved surprisingly more expensive, despite shorter initial hospital and readmission stays. Conclusion:Despite recent improvement in the success of IIT, WOP provides a more reliable and durable restoration of normoglycemia. Although IIT was associated with less procedure-related morbidity and shorter hospital stays, we unexpectedly found IIT to be more costly than WOP. This was largely due to IIT requiring islets from multiple donors to gain insulin independence. Because donor pancreata that are unsuitable for WOP can often be used successfully for IIT, we suggest that as IIT evolves, it should continue to be evaluated as a complementary alternative to rather than as a replacement for the better-established method of WOP.

Genome-wide analysis of histone modifications in human pancreatic islets

The global diabetes epidemic poses a major challenge. Epigenetic events contribute to the etiology of diabetes; however, the lack of epigenomic analysis has limited the elucidation of the mechanistic basis for this link. To determine the epigenetic architecture of human pancreatic islets we mapped the genome-wide locations of four histone marks: three associated with gene activation-H3K4me1, H3K4me2, and H3K4me3-and one associated with gene repression, H3K27me3. Interestingly, the promoters of the highly transcribed insulin and glucagon genes are occupied only sparsely by H3K4me2 and H3K4me3. Globally, we identified important relationships between promoter structure, histone modification, and gene expression. We demonstrated co-occurrences of histone modifications including bivalent marks in mature islets. Furthermore, we found a set of promoters that is differentially modified between islets and other cell types. We also use our histone marks to determine which of the known diabetes-associated single-nucleotide polymorphisms are likely to be part of regulatory elements. Our global map of histone marks will serve as an important resource for understanding the epigenetic basis of type 2 diabetes.

Calcineurin Signaling Regulates Human Islet β-Cell Survival

The calcium-regulated phosphatase calcineurin intersects with both calcium and cAMP-mediated signaling pathways in the pancreatic β-cell. Pharmacologic calcineurin inhibition, necessary to prevent rejection in the setting of organ transplantation, is associated with post-transplant β-cell failure. We sought to determine the effect of calcineurin inhibition on β-cell replication and survival in rodents and in isolated human islets. Further, we assessed whether the GLP-1 receptor agonist and cAMP stimulus, exendin-4 (Ex-4), could rescue β-cell replication and survival following calcineurin inhibition. Following treatment with the calcineurin inhibitor tacrolimus, human β-cell apoptosis was significantly increased. Although we detected no human β-cell replication, tacrolimus significantly decreased rodent β-cell replication. Ex-4 nearly normalized both human β-cell survival and rodent β-cell replication when co-administered with tacrolimus. We found that tacrolimus decreased Akt phosphorylation, suggesting that calcineurin could regulate replication and survival via the PI3K/Akt pathway. We identify insulin receptor substrate-2 (Irs2), a known cAMP-responsive element-binding protein target and upstream regulator of the PI3K/Akt pathway, as a novel calcineurin target in β-cells. Irs2 mRNA and protein are decreased by calcineurin inhibition in both rodent and human islets. The effect of calcineurin on Irs2 expression is mediated at least in part through the nuclear factor of activated T-cells (NFAT), as NFAT occupied the Irs2 promoter in a calcineurin-sensitive manner. Ex-4 restored Irs2 expression in tacrolimus-treated rodent and human islets nearly to baseline. These findings reveal calcineurin as a regulator of human β-cell survival in part through regulation of Irs2, with implications for the pathogenesis and treatment of diabetes following organ transplantation.

Single-Cell Transcriptomics of the Human Endocrine Pancreas.

Human pancreatic islets consist of multiple endocrine cell types. To facilitate the detection of rare cellular states and uncover population heterogeneity, we performed single-cell RNA sequencing (RNA-seq) on islets from multiple deceased organ donors, including children, healthy adults, and individuals with type 1 or type 2 diabetes. We developed a robust computational biology framework for cell type annotation. Using this framework, we show that α- and β-cells from children exhibit less well-defined gene signatures than those in adults. Remarkably, α- and β-cells from donors with type 2 diabetes have expression profiles with features seen in children, indicating a partial dedifferentiation process. We also examined a naturally proliferating α-cell from a healthy adult, for which pathway analysis indicated activation of the cell cycle and repression of checkpoint control pathways. Importantly, this replicating α-cell exhibited activated Sonic hedgehog signaling, a pathway not previously known to contribute to human α-cell proliferation. Our study highlights the power of single-cell RNA-seq and provides a stepping stone for future explorations of cellular heterogeneity in pancreatic endocrine cells.

Islet product characteristics and factors related to successful human islet transplantation from the collaborative islet transplant registry (CITR) 1999-2010

The Collaborative Islet Transplant Registry (CITR) collects data on clinical islet isolations and transplants. This retrospective report analyzed 1017 islet isolation procedures performed for 537 recipients of allogeneic clinical islet transplantation in 1999–2010. This study describes changes in donor and islet isolation variables by era and factors associated with quantity and quality of final islet products. Donor body weight and BMI increased significantly over the period (p < 0.001). Islet yield measures have improved with time including islet equivalent (IEQ)/particle ratio and IEQs infused. The average dose of islets infused significantly increased in the era of 2007–2010 when compared to 1999–2002 (445.4 ± 156.8 vs. 421.3 ± 155.4 ×103 IEQ; p < 0.05). Islet purity and total number of β cells significantly improved over the study period (p < 0.01 and <0.05, respectively). Otherwise, the quality of clinical islets has remained consistently very high through this period, and differs substantially from nonclinical islets. In multivariate analysis of all recipient, donor and islet factors, and medical management factors, the only islet product characteristic that correlated with clinical outcomes was total IEQs infused. This analysis shows improvements in both quantity and some quality criteria of clinical islets produced over 1999–2010, and these parallel improvements in clinical outcomes over the same period.