Gerace, D, Martiniello-Wilks, R, Habib, R, Ren, B, Nassif, NT, O'Brien, BA & Simpson, AM 2019, 'Ex Vivo Expansion of Murine MSC Impairs Transcription Factor-Induced Differentiation into Pancreatic β-Cells.', Stem Cells International, vol. 2019.View/Download from: Publisher's site
Combinatorial gene and cell therapy as a means of generating surrogate β-cells has been investigated for the treatment of type 1 diabetes (T1D) for a number of years with varying success. One of the limitations of current cell therapies for T1D is the inability to generate sufficient quantities of functional transplantable insulin-producing cells. Due to their impressive immunomodulatory properties, in addition to their ease of expansion and genetic modification ex vivo, mesenchymal stem cells (MSCs) are an attractive alternative source of adult stem cells for regenerative medicine. To overcome the aforementioned limitation of current therapies, we assessed the utility of ex vivo expanded bone marrow-derived murine MSCs for their persistence in immune-competent and immune-deficient animal models and their ability to differentiate into surrogate β-cells. CD45-/Ly6+ murine MSCs were isolated from the bone marrow of nonobese diabetic (NOD) mice and nucleofected to express the bioluminescent protein, Firefly luciferase (Luc2). The persistence of a subcutaneous (s.c.) transplant of Luc2-expressing MSCs was assessed in immune-competent (NOD) (n = 4) and immune-deficient (NOD/Scid) (n = 4) animal models of diabetes. Luc2-expressing MSCs persisted for 2 and 12 weeks, respectively, in NOD and NOD/Scid mice. Ex vivo expanded MSCs were transduced with the HMD lentiviral vector (MOI = 10) to express furin-cleavable human insulin (INS-FUR) and murine NeuroD1 and Pdx1. This was followed by the characterization of pancreatic transdifferentiation via reverse transcriptase polymerase chain reaction (RT-PCR) and static and glucose-stimulated insulin secretion (GSIS). INS-FUR-expressing MSCs were assessed for their ability to reverse diabetes after transplantation into streptozotocin- (STZ-) diabetic NOD/Scid mice (n = 5). Transduced MSCs did not undergo pancreatic transdifferentiation, as determined by RT-PCR analyses, lacked glucose responsiveness, and upon transplantation did no...
Ren, B, La, QT, O'Brien, BA, Nassif, NT, Tan, Y, Gerace, D, Martiniello-Wilks, R, Torpy, F, Dane, AP, Alexander, IE & Simpson, AM 2018, 'Partial pancreatic transdifferentiation of primary human hepatocytes in the livers of a humanised mouse model.', The journal of gene medicine, vol. 20, no. 5.View/Download from: Publisher's site
Gene therapy is one treatment that may ultimately cure type 1 diabetes. We have previously shown that the introduction of furin-cleavable human insulin (INS-FUR) to the livers in several animal models of diabetes resulted in the reversal of diabetes and partial pancreatic transdifferentiation of liver cells. The present study investigated whether streptozotocin-diabetes could be reversed in FRG mice in which chimeric mouse-human livers can readily be established and, in addition, whether pancreatic transdifferentiation occurred in the engrafted human hepatocytes.Engraftment of human hepatocytes was confirmed by measuring human albumin levels. Following delivery of the empty vector or the INS-FUR vector to diabetic FRG mice, mice were monitored for weight and blood glucose levels. Intraperitoneal glucose tolerance tests (IPGTTs) were performed. Expression levels of pancreatic hormones and transcription factors were determined by a reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry.Diabetes was reversed for a period of 60 days (experimental endpoint) after transduction with INS-FUR. IPGTTs of the insulin-transduced animals were not significantly different from nondiabetic animals. Immunofluorescence microscopy revealed the expression of human albumin and insulin in transduced liver samples. Quantitative RT-PCR showed expression of human and mouse endocrine hormones and β-cell transcription factors, indicating partial pancreatic transdifferentiation of mouse and human hepatocytes. Nonfasting human C-peptide levels were significantly higher than mouse levels, suggesting that transdifferentiated human hepatocytes made a significant contribution to the reversal of diabetes.These data show that human hepatocytes can be induced to undergo partial pancreatic transdifferentiation in vivo, indicating that the technology holds promise for the treatment of type 1 diabetes.
Ren, B, Tao, C, Swan, MA, Joachim, N, Martiniello-Wilks, R, Nassif, NT, O'Brien, BA & Simpson, AM 2016, 'Pancreatic Transdifferentiation and Glucose-Regulated Production of Human Insulin in the H4IIE Rat Liver Cell Line', INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 17, no. 4.View/Download from: Publisher's site
Lawandi, J, Tao, C, Ren, B, Williams, P, Ling, D, Swan, MA, Nassif, NT, Torpy, FR, O'Brien, BA & Simpson, AM 2015, 'Reversal of diabetes following transplantation of an insulin-secreting human liver cell line: Melligen cells', MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT, vol. 2.View/Download from: Publisher's site
Gerace, D, Ren, B, Hawthorne, W, Byrne, M, Phillips, P, O'Brien, B, Nassif, N, Alexander, I & Simpson, AM 2013, 'Pancreatic transdifferentiation in porcine liver following lentiviral delivery of human furin-cleavable insulin', Transplantation Proceedings, vol. 45, no. 5, pp. 1869-1874.View/Download from: Publisher's site
Type I diabetes mellitus (TID) results from the autoimmune destruction of the insulin-producing pancreatic ß-cells. Gene therapy is one strategy being actively explored to cure TID by affording non-ß-cells the ability to secrete insulin in response to physiologic stimuli. In previous studies, we used a novel surgical technique to express furin-cleavable human insulin (INS-FUR) in the livers of streptozotocin (STZ)-diabetic Wistar rats and nonobese diabetic (NOD) mice with the use of the HMD lentiviral vector. Normoglycemia was observed for 500 and 150 days, respectively (experimental end points). Additionally, some endocrine transdifferentiation of the liver, with storage of insulin in granules, and expression of some ß-cell transcription factors (eg, Pdx1, Neurod1, Neurog3, Nkx2-2, Pax4) and pancreatic hormones in both studies. The aim of this study was to determine if this novel approach could induce liver to pancreatic transdifferentiation to reverse diabetes in pancreatectomized Westran pigs. Nine pigs were used in the study, however only one pig maintained normal fasting blood glucose levels for the period from 10 to 44 days (experimental end point). This animal was given 2.8 × 10(9) transducing units/kg of the lentiviral vector expressing INS-FUR. A normal intravenous glucose tolerance test was achieved at 30 days. Reverse-transcription polymerase chain reaction analysis of the liver tissue revealed expression of several ß-cell transcription factors, including the key factors, Pdx-1 and Neurod1, pancreatic hormones, glucagon, and somatostatin; however, endogenous pig insulin was not expressed.
Ren, B, O'Brien, B, Byrne, M, Ch'ng, E, Gatt, PN, Swan, MA, Nassif, N, Wei, M, Gijsbers, R, Debyser, Z & Simpson, AM 2013, 'Long-term reversal of diabetes in non-obese diabetic mice by liver-directed gene therapy.', The Journal of gene Medicine, vol. 15, no. 1, pp. 28-41.View/Download from: Publisher's site
Background Type 1 diabetes (T1D) results from an autoimmune attack against the insulin-producing ß-cells of the pancreas. The present study aimed to reverse T1D by gene therapy. Methods We used a novel surgical technique, which involves isolating the liver from the circulation before the delivery of a lentiviral vector carrying furin-cleavable human insulin (INS-FUR) or empty vector to the livers of diabetic non-obese diabetic mice (NOD). This was compared with the direct injection of the vector into the portal circulation. Mice were monitored for body weight and blood glucose. Intravenous glucose tolerance tests were performed. Expression of insulin and pancreatic transcription factors was determined by the reverse transcriptase-polymerase chain reaction and immunohistochemistry and immunoelectron microscopy was used to localise insulin. Results Using the novel surgical technique, we achieved long-term transduction (42% efficiency) of hepatocytes, restored normoglycaemia for 150 days (experimental endpoint) and re-established normal glucose tolerance. We showed the expression of ß-cell transcription factors, murine insulin, glucagon and somatostatin, and hepatic storage of insulin in granules. The expression of hepatic markers, C/EBP-ß, G6PC, AAT and GLUI was down-regulated in INS-FUR-treated livers. Liver function tests remained normal, with no evidence of intrahepatic inflammation or autoimmune destruction of the insulin-secreting liver tissue. By comparison, direct injection of INS-FUR reduced blood glucose levels, and no pancreatic transdifferentiation or normal glucose tolerance was observed.
Simpson, AM, Ren, B, O'Brien, BA & Nassif, NT 2013, 'Response to the letter to the editor by M. Elsner et al: "Comment on Binhai Ren et al (2013;15:28-41). Long term reversal of diabetes in non-obese diabetic mice by liver-directed gene therapy"', JOURNAL OF GENE MEDICINE, vol. 15, no. 8-9, pp. 309-310.View/Download from: Publisher's site
Mccann, L, Haywood, M, Ren, B, Simpson, AM, Guilhaus, M, Wasinger, V, Raftery, MJ & Davey, RA 2007, 'Identification Of Vascular Surface Proteins By In Vivo Biotinylation: A Method Sufficiently Sensitive To Detect Changes In Rat Liver 2 Weeks After Partial Hepatectomy', Journal Of Proteome Research, vol. 6, no. 8, pp. 3108-3113.View/Download from: Publisher's site
We have developed a methodology to selectively isolate and identify proteins associated with the luminal surface of blood vessels using in vivo biotinylation, streptavidin-affinity chromatography, and SDS-PAGE/LC-MS/MS. This had sufficient sensitivity to
Ren, B, O'Brien, B, Swan, MA, Koina, ME, Nassif, N, Wei, MQ & Simpson, AM 2007, 'Long-term Correction Of Diabetes In Rats After Lentiviral Hepatic Insulin Gene Therapy', Diabetologia, vol. 50, no. 9, pp. 1910-1920.View/Download from: Publisher's site
Aims/hypothesis Type 1 diabetes results from the autoimmune destruction of pancreatic beta cells. Exogenous insulin therapy cannot achieve precise physiological control of blood glucose concentrations, and debilitating complications develop. Lentiviral v
Ren, B, O'Brien, B, Swan, MA & Simpson, AM 2005, 'In vivo delivery of the human insulin gene results in long-term reversal of streptozotocin-induced type 1 diabetes in rats', Journal Of Gene Medicine, vol. 7, no. 8, pp. 1124-1124.
Gerace, D, Ren, B, Martiniello-Wilks, R & Simpson, AM 2019, 'High-Efficiency Lentiviral Gene Modification of Primary Murine Bone-Marrow Mesenchymal Stem Cells.' in Progenitor Cells: Methods and Protocols, Springer, Switzerland, pp. 197-214.View/Download from: Publisher's site
Lentiviral vectors are the method of choice for stable gene modification of a variety of cell types. However, the efficiency with which they transduce target cells varies significantly, in particular their typically poor capacity to transduce primary stem cells. Here we describe the isolation and enrichment of murine bone-marrow mesenchymal stem cells (MSCs) via fluorescence-activated cell sorting (FACS); the cloning, production, and concentration of high-titer second generation lentiviral vectors via combined tangential flow filtration (TFF) and ultracentrifugation; and the subsequent high-efficiency gene modification of MSCs into insulin-producing cells via overexpression of the furin-cleavable human insulin (INS-FUR) gene.
Ren, B, La, QT, O'Brien, BA, Nassif, NT, Tan, Y, Gerace, D, Martiniello-Wilks, R, Torpy, F, Dane, AP, Alexander, IE & Simpson, AM 2017, 'EXPRESSION OF HUMAN PANCREATIC TRANSCRIPTION FACTORS IN THE LIVERS OF FRG MICE', JOURNAL OF GENE MEDICINE, Joint 10th Annual Scientific Meeting of the Australian-Gene-and-Cell-Therapy-Society (AGCTS) and Australasian-Society-for-Stem-Cell-Research (ASSCR), WILEY, Univ Technol Sydney, Sydney, AUSTRALIA.
Gerace, D, Martiniello-Wilks, R, Nassif, NT, Ren, B & Simpson, AM 2017, 'Ex Vivo Expanded Murine Mesenchymal Stem Cells as Targets for the Generation of a Cell Replacement Therapy for Type 1 Diabetes', DIABETES, 77th Scientific Sessions of the American-Diabetes-Association, AMER DIABETES ASSOC, San Diego, CA, pp. A83-A83.
Ren, B, O'Brien, BA, Alexander, IE, Nassif, NT, Tan, Y, Martiniello-Wilks, R & Simpson, AM 2015, 'Pancreatic Transdiffereniation of Human Hepatocytes in the Livers of a Humanized Mouse Model', MOLECULAR THERAPY, 18th Annual Meeting of the American-Society-of-Gene-and-Cell-Therapy (ASGCT), NATURE PUBLISHING GROUP, New Orleans, LA, pp. S110-S110.View/Download from: Publisher's site
Simpson, A, Ren, B, O'Brien, BA, Alexander, IE, Nassif, NT, Tan, Y & Martiniello-Wilks, R 2015, 'Gene therapy for diabetes: reversal of diabetes in the humanised FRG mouse model', XENOTRANSPLANTATION, IPITA/IXA/CTS Joint Congress, WILEY-BLACKWELL, Melbourne, AUSTRALIA, pp. S41-S42.
Simpson, A, Ren, B, O'Brien, BA, Alexander, IE, Nassif, NT, Tan, Y & Martiniello-Wilks, R 2015, 'GENE THERAPY FOR DIABETES: REVERSAL OF DIABETES IN THE HUMANISED FRG MOUSE MODEL.', TRANSPLANTATION, Joint Congress of the International-Pancreas-and-Islet-Transplantation-Association, International-Xenotransplantation-Association and Cell-Transplant-Society, LIPPINCOTT WILLIAMS & WILKINS, Melbourne, AUSTRALIA, pp. S67-S67.
Lawandi, J, Tao, C, Ren, B, Williams, P, Ling, D, Swan, MA, Nassif, NT, Torpy, FR, O'Brien, BA & Simpson, AM 2015, 'MELLIGEN CELSS: AN INSULIN-SECRETING HUMAN LIVER CELL LINE WHICH IS RESISTANT TO CYTOKINE-INDUCED IMMUNE ATTACK', JOURNAL OF GENE MEDICINE, 9th Biennial Meeting of the Australasian-Gene-and-Cell-Therapy-Society (AGCTS), WILEY-BLACKWELL, Univ Melbourne, Univ Coll, Parkville, AUSTRALIA, pp. 189-189.
Gerace, D, Ren, B, Hawthorne, WJ, Byrne, MR, Phillips, P, O'Brien, BA, Nassif, N, Alexander, IE & Simpson, AM 2013, 'REVERSAL OF DIABETES IN A PORCINE MODEL FOLLOWING LIVER-DIRECTED GENE THERAPY', JOURNAL OF GENE MEDICINE, 8th Meeting of the Australasian-Gene-Therapy-Society, WILEY-BLACKWELL, Univ Technol, Sydney, AUSTRALIA, pp. 326-326.