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

Thesis (PhD)--Stellenbosch University, 2017ENGLISH 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 patient’s 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+ similar to the endogenous islets in the pancreas. In conclusion, MSCs promote the expansion of Pdx1+ cells and maintain the expression of Ngn3 in islet cells and duct–derived neogenetic cells. MSCs prolong graft survival and improve the capacity of early harvested post PDL islets to reverse hyperglycemia; this novel observation may be applicable to clinical transplantation.AFRIKAANSE OPSOMMING : 'n Tekort aan skenker eilandjies, immuun verwerping, sowel as die behoefte aan lewenslange immuno-onderdrukkers is van die grootste kliniese uitdagings in eiland oorplanting in die behandeling van diabetes mellitus. 'n Alternatief tot hierdie uitdagings is die in vivo generering van betaselle in die pankreas van die pasiënt. 'n Dier model vir pankreas beserings is voorheen berig as 'n potensiële bron van eiland selle vir weefsel vervangingsterapie in tipe 1-diabetes mellitus, maar verdere ondersoek met betrekking tot die in vitro regeneratiewe kapasiteit van endogene betaselle in hierdie model is nodig. Hierdie studie ondersoek, in vitro, die effek van post-pankreatiese buis afbinding (PPBA) - gëinduseerde eiland / buis-mesenkiemale stroma sel (MSS) interaksies op eiland en buis sel ontwikkeling en beoordeel die langtermyn oorplanting uitkoms van eiland-mesenkiemale selle. Eilandjies, buis fragmente, en MSS is geïsoleer vanaf tagtig volwasse mannetjie Wistar rotte (250 - 300g) en geoes 24- en 120- ure nadat buis afbinding voltooi is. Eilandjies of buis fragmente was gekweek in die aan- of af-wesigheid van MSS (eiland / MSS + of eiland / MSS -). Die ontwikkeling van eilandjie en buis fragmente in kultuur, is met behulp van morfologie asook die aanwesigheid van teenliggaampies vir Pdx1, Ngn3, CK7 en insulien, wat deur immunositochemie evalueer is. Eilandjies is ook in diabeet rotte (n = 40) oorgeplant in in die aan- of af - wesigheid van MSS (eiland / MSS + of eiland / MSS -). Oorplantingsoorlewing en funksie is evalueer deur bloed glukose vlakke te asseseer en ent weefsel immunohistochemie te bestudeer. Resultate het die aktivering van Pdx1+ eiland selle in beide die aan- of afwesigheid van MSS aangetoon. Uitbreiding van Pdx1+ selle is bevorder in die aanwesigheid van mss wat gevolg is deur 'aktivering van Ngn3 uitdrukking asook uitbreiding van Ngn3+ selle, wat in eiland selle is in stand gehou is tot en met vier weke. Dit het gelei tot lae vlakke van insulien uitdrukking in eiland-agtige aggregate wat gevorm het tussen die 3de en die 4de week. Medekweking van buis fragmente met MSS het tot die instandhouding van endokriene voorlopers wat Ngn3 uit druk, en later eilandagtige aggregate gevorm het, tot gevolg gehad. In kulture met MSS, ontwikkel buis epiteelsellle groeigebiede met selle wat beide CK7 en Ngn3 uitdruk in peribuis selle. Nadat peribuis selle eiland-agtige aggregate vorm, is Ngn3 saam met insulien uitgedruk binne eiland-agtige sel trosse naastens aan buisies. Oorplanting van vroeë oes (24 uur PPBA) eilandjies het beter genesende vermoë as laat (84 uur PPBA) eilandjies getoon. Die gemiddelde glucose vlakke was laer deur die 5 weke-lange moniteringstydperk in rotte waarin oorplanting 24 uur PPBA uitgerig is. Die gemiddelde tyd om hiperglisemie om te keer in 80% van die 24 uur PPBA oorplanting groep was 32 ± 2 dae (~ 4.5 weke), terwyl slegs 20% in die 84 uur PPBA oorplanting groep normoglisemie bereik het op 61 ± 2 dae (~ 9 weke) (p = 0,0011) na oorplanting. Ent oorlewing was hoër in eilandjies wat in die teenwoordigheid van MSS (eiland / MSS +) in vergelyking met ente wat oorplant is met eilandjies alleen (eiland / MSS -). Eilandjie morfologie en verspreiding van betaselle was normaal in eiland / MSS+, soortgelyk aan die endogene eilandjies in die pankreas. Ten slotte, MSS bevorder die uitbreiding van Pdx1+ selle en handhaaf die uitdrukking van Ngn3 in eiland selle en-buis afgeleide neogenetiese sell. MSS verleng ent oorlewing en verbeter die kapasiteit van die vroeë oes PPBA eilandjies om hyperglisemie om te keer; hierdie waarneming mag van toepassing wees op kliniese oorplanting