Progress in individualizing autologous islet isolation techniques for pediatric islet autotransplantation after total pancreatectomy in children for chronic pancreatitis.

Total pancreatectomy with islet autotransplantation is performed to treat chronic pancreatitis in children. Successful islet isolation must address the challenges of severe pancreatic fibrosis and young donor age. We have progressively introduced modifications to optimize enzymatic and mechanical dissociation of the pancreas during islet isolation. We evaluated 2 islet isolation metrics in 138 children-digest islet equivalents per gram pancreas tissue (IEQ/g) and digest IEQ per kilogram body weight (IEQ/kg), using multiple regression to adjust for key disease and patient features. Islet yield at digest had an average 4569 (standard deviation 2949) islet equivalent (IEQ)/g and 4946 (4009) IEQ/kg, with 59.1% embedded in exocrine tissue. Cases with very low yield (<2000 IEQ/g or IEQ/kg) have decreased substantially over time, 6.8% and 9.1%, respectively, in the most recent tertile of time compared to 19.2% and 23.4% in the middle and 34.1% and 36.4% in the oldest tertile. IEQ/g and IEQ/kg adjusted for patient and disease factors improved in consistency and yield in the modern era. Minimal mechanical disruption during digestion, warm enzymatic digestion using enzyme collagenase:NP activity ratio < 10:1, coupled with extended distension and trimming time during islet isolation of younger and fibrotic pediatric pancreases, gave increased islet yield with improved patient outcomes.

HLA-G1+ Expression in GGTA1KO Pigs Suppresses Human and Monkey Anti-Pig T, B and NK Cell Responses.

The human leukocyte antigen G1 (HLA-G1), a non-classical class I major histocompatibility complex (MHC-I) protein, is a potent immunomodulatory molecule at the maternal/fetal interface and other environments to regulate the cellular immune response. We created GGTA1-/HLAG1+ pigs to explore their use as organ and cell donors that may extend xenograft survival and function in both preclinical nonhuman primate (NHP) models and future clinical trials. In the present study, HLA-G1 was expressed from the porcine ROSA26 locus by homology directed repair (HDR) mediated knock-in (KI) with simultaneous deletion of α-1-3-galactotransferase gene (GGTA1; GTKO) using the clustered regularly interspersed palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) (CRISPR/Cas9) gene-editing system. GTKO/HLAG1+ pigs showing immune inhibitory functions were generated through somatic cell nuclear transfer (SCNT). The presence of HLA-G1 at the ROSA26 locus and the deletion of GGTA1 were confirmed by next generation sequencing (NGS) and Sanger's sequencing. Fibroblasts from piglets, biopsies from transplantable organs, and islets were positive for HLA-G1 expression by confocal microscopy, flow cytometry, or q-PCR. The expression of cell surface HLA-G1 molecule associated with endogenous ß2-microglobulin (ß2m) was confirmed by staining genetically engineered cells with fluorescently labeled recombinant ILT2 protein. Fibroblasts obtained from GTKO/HLAG1+ pigs were shown to modulate the immune response by lowering IFN-γ production by T cells and proliferation of CD4+ and CD8+ T cells, B cells and natural killer (NK) cells, as well as by augmenting phosphorylation of Src homology region 2 domain-containing phosphatase-2 (SHP-2), which plays a central role in immune suppression. Islets isolated from GTKO/HLA-G1+ genetically engineered pigs and transplanted into streptozotocin-diabetic nude mice restored normoglycemia, suggesting that the expression of HLA-G1 did not interfere with their ability to reverse diabetes. The findings presented here suggest that the HLA-G1+ transgene can be stably expressed from the ROSA26 locus of non-fetal maternal tissue at the cell surface. By providing an immunomodulatory signal, expression of HLA-G1+ may extend survival of porcine pancreatic islet and organ xenografts.

Mitochondrial Proton Leak Regulated by Cyclophilin D Elevates Insulin Secretion in Islets at Nonstimulatory Glucose Levels.

Fasting hyperinsulinemia precedes the development of type 2 diabetes. However, it is unclear whether fasting insulin hypersecretion is a primary driver of insulin resistance or a consequence of the progressive increase in fasting glycemia induced by insulin resistance in the prediabetic state. Herein, we have discovered a mechanism that specifically regulates non-glucose-stimulated insulin secretion (NGSIS) in pancreatic islets that is activated by nonesterified free fatty acids, the major fuel used by ß-cells during fasting. We show that the mitochondrial permeability transition pore regulator cyclophilin D (CypD) promotes NGSIS, but not glucose-stimulated insulin secretion, by increasing mitochondrial proton leak. Islets from prediabetic obese mice show significantly higher CypD-dependent proton leak and NGSIS compared with lean mice. Proton leak-mediated NGSIS is conserved in human islets and is stimulated by exposure to nonesterified free fatty acids at concentrations observed in obese subjects. Mechanistically, proton leak activates islet NGSIS independently of mitochondrial ATP synthesis but ultimately requires closure of the KATP channel. In summary, we have described a novel nonesterified free fatty acid-stimulated pathway that selectively drives pancreatic islet NGSIS, which may be therapeutically exploited as an alternative way to halt fasting hyperinsulinemia and the progression of type 2 diabetes.

Optimizing sgRNA length to improve target specificity and efficiency for the GGTA1 gene using the CRISPR/Cas9 gene editing system.

The CRISPR/Cas9 gene editing system has enhanced the development of genetically engineered animals for use in xenotransplantation. Potential limitations to the CRISPR/Cas9 system impacting the development of genetically engineered cells and animals include the creation of off-target mutations. We sought to develop a method to reduce the likelihood of off-target mutation while maintaining a high efficiency rate of desired genetic mutations for the GGTA1 gene. Extension of sgRNA length, responsible for recognition of the target DNA sequence for Cas9 cleavage, resulted in improved specificity for the GGTA1 gene and less off-target DNA cleavage. Three PAM sites were selected within exon 1 of the porcine GGTA1 gene and ten sgRNA of variable lengths were designed across these three sites. The sgRNA was tested against synthetic double stranded DNA templates replicating both the native GGTA1 DNA template and the two most likely off-target binding sites in the porcine genome. Cleavage ability for native and off-target DNA was determined by in vitro cleavage assays. Resulting cleavage products were analyzed to determine the cleavage efficiency of the Cas9/sgRNA complex. Extension of sgRNA length did not have a statistical impact on the specificity of the Cas9/sgRNA complex for PAM1 and PAM2 sites. At the PAM3 site, however, an observed increase in specificity for native versus off-target templates was seen with increased sgRNA length. In addition, distance between PAM site and the start codon had a significant impact on cleavage efficiency and target specificity, regardless of sgRNA length. Although the in vitro assays showed off-target cleavage, Sanger sequencing revealed that no off-target mutations were found in GGTA1 knockout cell lines or piglet. These results demonstrate an optimized method for improvement of the CRIPSR/Cas9 gene editing system by reducing the likelihood of damaging off-target mutations in GGTA1 knocked out cells destined for xenotransplant donor production.

Individual islet respirometry reveals functional diversity within the islet population of mice and human donors.

OBJECTIVE: Islets from the same pancreas show remarkable variability in glucose sensitivity. While mitochondrial respiration is essential for glucose-stimulated insulin secretion, little is known regarding heterogeneity in mitochondrial function at the individual islet level. This is due in part to a lack of high-throughput and non-invasive methods for detecting single islet function. METHODS: We have developed a novel non-invasive, high-throughput methodology capable of assessing mitochondrial respiration in large-sized individual islets using the XF96 analyzer (Agilent Technologies). RESULTS: By increasing measurement sensitivity, we have reduced the minimal size of mouse and human islets needed to assess mitochondrial respiration to single large islets of >35,000 µm2 area (∼210 µm diameter). In addition, we have measured heterogeneous glucose-stimulated mitochondrial respiration among individual human and mouse islets from the same pancreas, allowing population analyses of islet mitochondrial function for the first time. CONCLUSIONS: We have developed a novel methodology capable of analyzing mitochondrial function in large-sized individual islets. By highlighting islet functional heterogeneity, we hope this methodology can significantly advance islet research.