Abstracts Category : Other

An in vitro study of mesenchymeislet cell interactions in islet neogenesis: A model for tissue replacement therapy in diabetes mellitus

by Juziel Kampando Manda

ENGLISH ABSTRACT : Shortages of donor islets, immune rejection, and the need for life-long immuno-suppressors remain the clinical challenges of islet transplantation in the treatment of diabetes mellitus. An alternative to these challenges is the in vivo generation of beta cells within the patients pancreas. The animal model of pancreatic injury has been reported to be a potential source of islet cells for tissue replacement therapy in type 1 diabetes mellitus. However, the in vitro regenerative capacity of endogenous beta cells in this model needs more investigation. This study investigated, in vitro, the effect of pancreatic duct ligation (PDL)-induced islet/duct-mesenchymal stromal cells (MSCs) interactions on islet and duct cells development and assessed the long-term transplantation outcome of islet-mesenchymal cells isografts. Islets, duct fragments, and MSCs were isolated from post PDL tissues harvested from eighty adult male Wistar rats (250 - 300g) 24- and 120 h following duct ligation. Islets or duct fragments were cultured with or without MSCs ([Islet/MSC+ or Islet/MSC-] or [PEDC/MSC+ or PEDC/MSC-]). Development of islets and duct fragments in culture were evaluated morphologically and by immunocytochemistry using antibodies against Pdx1, Ngn3, CK7 and insulin. Islets were also transplanted with or without MSCs (Islet/MSC+ or islet/MSC-) in diabetic animals (n = 40). Isografts survival and function were evaluated by monitoring blood glucose levels, and immunohistochemistry of graft tissues were studied. Results showed activation of Pdx1+ islet cells in both cultures with or without MSCs, however, expansion of Pdx1+ cells were promoted in the presence of MSCs and this was followed by activation of Ngn3 expression and expansion of Ngn3+ cells, which was maintained in islet cells up to 4 weeks. This resulted into low levels of insulin expression in islet-like aggregates formed between the third and the fourth week. Co-culturing of duct fragments with MSC similarly resulted into maintenance of endocrine precursors that expressed Ngn3, which later formed islet-like aggregates. In cultures with MSCs, duct epithelial cells developed growth areas with cells that co-expressed CK7 and Ngn3 in periductal cells. When periductal cells formed islet-like aggregates, Ngn3 co-expressed with insulin in islet-like cell clusters closer to ducts. Transplantation of early harvested (24 h PPDL) islets showed better curative capacity than late (84 h PPDL) islets. The average glucose levels were lower throughout the 5 weeks monitoring period in 24 h PPDL transplanted rats. The average time to reverse hyperglycemia in 80% of the 24 h PPDL transplant group was 32 2 days (~4.5 weeks), while only 20% in the 84 h PPDL transplant group attained normoglycemia at 61 2 days (~9 weeks) (p = 0.0011) post transplantation. Graft survival rate was higher in islets co-transplanted with MSC (Islet/MSC+) compared to grafts transplanted with islets alone (Islet/MSC-). Islet morphology and distribution of beta cells was normal in Islet/MSC+Advisors/Committee Members: Tchokonte-Nana, Venant, Page, Benedict John, Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Anatomy and Histology.