Chair for Molecular Animal Breeding and Biotechnology, Gene Center
INS-eGFP transgenic piglets provide a novel tool for studying maturation and vascularization of neonatal islet cell clusters (NICCs) in vivo
Elisabeth Kemter1, Christian Cohrs2,3,4, Andrea Wolf5, Annegret Wuensch1, Mayuko Kurome1, Barbara Kessler1, Valeri Zakhartchenko1, Matthias Loehn5, Yuri Ivashchenko5, Stephan Speier2,3,4, Anke Schulte5, Eckhard Wolf1.
1Chair for Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, Munich, Germany; 2Paul Langerhans Institute Dresden (PLID) at the University Clinic Carl Gustav Carus of TU Dresden, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany; 3German Centre for Diabetes Research (DZD), Dresden, Germany; 4DFG-Center for Regenerative Therapies Dresden (CRTD), , Faculty of Medicine, Technische Universität Dresden, Dresden, Germany; 5Diabetes Division, Sanofi-Aventis, Frankfurt, Germany
Porcine neonatal islet cell clusters (NICCs) are an attractive source of beta-cells for xenotransplantation into diabetic recipients. The isolation of NICCs is well established and standardized. Furthermore, the procedure is technically and financially less demanding and more robust than islet isolation from adult donor pigs. NICCs have the capacity to proliferate, increasing their cell mass after transplantation. However, freshly isolated NICCs are not fully functional and require maturation in vitro or - after transplantation - in vivo until beta-cells secrete enough insulin to be therapeutically effective. The choice of the optimal transplantation site and the vascularization of NICCs after transplantation are other issues to be solved.
To address these points, we generated transgenic pigs expressing eGFP under the control of the porcine insulin promoter in the pancreatic beta-cells. Porcine kidney cells were transfected with a porcine INS promoter-eGFP expression vector, pools of stable transfected cell clones were used for somatic cell nuclear transfer (SCNT), and cloned embryos were transferred to recipient gilts. Offspring were genotyped by PCR and Southern blot analysis and characterized for reporter gene expression. NICCs were isolated using a standard procedure. Briefly, whole pancreata of piglets (< 8 days old) were minced in small pieces and shortly digested with collagenase. The suspension was sieved through a 500-µm mesh and exocrine cells were removed within the first three days in culture. The best culture conditions proved to be recovery media of Ham’s F12/M199 with additives of protease inhibitors, antioxidants and additional nutrients for the first three post-isolation days followed by maturation media of Ham’s F10 with IBMX. After in vitro maturation, NICCs were characterized by immunofluorescence analysis, glucose stimulated insulin secretion (GSIS) assays, and beta-cell specific proliferation assays.
A total of 12 live INS-eGFP transgenic founder piglets were born, with a total of 10 different transgene integration patterns. Fluorescence microscopy and immunohistochemistry for GFP in sections of pancreas and a panel of other tissues demonstrated islet-specific transgene expression. Consistent with these results, isolated NICCs of the transgenic founder pigs showed different levels of eGFP fluorescence (1 strong, 1 weak, all others intermediate). Double-immunofluorescence analysis identified eGFP expression specifically in the beta-cells; eGFP was neither detected in glucagon positive alpha-cells nor in somatostatin positive delta-cells. Two founders with high and intermediate beta-cell specific eGFP expression were successfully reproduced by SCNT to set up breeding lines of donor pigs for large-scale NICC isolation. To observe their maturation in vivo, NICCs from the pig line with intermediate eGFP expression were transplanted into the anterior eye chamber of the NODscid mouse and regularly monitored by in vivo fluorescence microscopy. While 3 days after transplantation eGFP expression of the NICCs was weak, it increased markedly by day 14 and even more by day 28 post transplantation. The NICC mass increased several-fold during this period and a dense vascular network was formed in the transplanted NICCs. Future studies will use this system to optimize the conditions for maturation and vascularization of NICCs after xenotransplantation.
Funded by Sanofi and by the Deutsche Forschungsgemeinschaft (TR-CRC 127).
11:00 - 12:30
|Alternative Sources of Beta Cells: Xenoislet, Stem Cells, Tissue Engineering||INS-eGFP transgenic piglets provide a novel tool for studying maturation and vascularization of neonatal islet cell clusters (NICCs) in vivo||Room 110|