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Generation of pancreatic beta-cells from embryonic stem cells

Mattias Hansson  


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Accepted by: Faculty of Health Sciences University of Copenhagen
Defended on: December 12, 2005
Official opponents: Hanne Cathrine Bisgaard , Jens Zimmer Rasmussen , Lars Wahlberg
Tutors: Palle Serup , Jens Høiriis Nielsen

Published in the PhD Database: July 11, 2006


English abstract
Diabetes mellitus is a huge public health problem. The common treatment of type 1 diabetes (T1D) relies on insulin injections, which is cumbersome and associated with severe vascular complications leading to blindness, kidney failure, and amputations. Today, the cure of T1D depends on the replacement of the β-cell mass by transplantation of pancreas or islet cells. The transplantation of islets can restore normoglycemia and insulin independence without massive surgery, which reduces the risk of surgical complications as well as the cost. As with all transplantation therapies, this treatment is hampered by a lack of suitable donors. Recent progress in stem cell research raises the hope of enabling re-establishment of a functional β-cell mass by transplantation of in vitro generated β-cells. The use of embryonic stem cells (ESCs) might provide an unlimited source of material for transplantation in the future. ESCs are pluripotent cells that can be cultured indefinitely in an undifferentiated state. However, these cells can be induced to differentiate into various cell types. In this report, the potential of ESCs to differentiate into definitive endodermal cell lineages, including pancreatic cell types is studied.
Recent studies have reported that insulin-producing cells can be generated from nestin-expressing neuronal progenitors derived from ESCs. The potential of these progenitor cells is further investigated in this study. Here, it is shown that nestin+ progenitors do not give rise to insulin-producing cells. The insulin found in ESC progeny is not due to insulin synthesis but rather due to an uptake of exogenous insulin from the cell culture medium. Many insulin+ cells are apoptotic and trap insulin from the medium.
Nodal, a member of the TGF-β superfamily, has been shown to play a pivotal role in the formation of endoderm in vertebrates. In this study, the role of Nodal signaling during differentiation of endoderm from ESCs is investigated. The results indicate that the Nodal pathway influences downstream endodermal gene expression. However, it is not possible by this analysis to fully distinguish the development of visceral and definitive endoderm.
ESCs can form benign tumors, teratomas, when injected in syngenic or immunocompromised mice. Here, the potential role of teratomas as an in vivo differentiation model of pancreatic cells from ESCs is evaluated. The initial analysis of ESC-derived teratomas suggests that gut-like endodermal epithelium is formed and the differentiation of pancreatic cells is partly confirmed.



Danish abstract
Patienter med type 1 diabetes (T1D) behandles almindeligvis ved hjælp af daglige insulin indsprøjtninger. Det er besværligt, og der opstår ofte alvorlige sekundære komplikationer på grund af kredsløbsforstyrrelser, der kan resultere i nyresvigt, amputationer og tab af synsevnen. Muligheden for helbredelse af T1D i dag beror på, at man erstatter de ødelagte β-celler ved hjælp af organtransplantation af bugspytkirtlen (pankreas) eller oprensede Langerhanske øer. Transplantation af ø-celler kan resultere i normalisering af blodsukkerreguleringen, og dermed gøre patienten uafhængig af insulin uden at skulle gennemføre et større operativt indgreb. Fordelen er, at det reducerer de økonomiske omkostninger samt risikoen for komplikationer i forhold til at transplantere hele pankreas. Desværre er der alt for få velegnede donororganer til rådighed. De nyeste resultater inden for stamcelleforskning giver håb om, at det vil blive muligt at genetablere en funktionel β-cellemasse i patienter ved at transplantere in vitro dyrkede β-celler. Ved at bruge embryonale stamceller (ESC) vil man potentielt have en ubegrænset ressource af transplantationsmateriale i fremtiden. ESC er pluripotente celler, som kan dyrkes og holdes i et udifferentieret stadium, men de kan også påvirkes til at differentiere til forskellige celletyper. I dette arbejde er undersøgt ESCs potentiale til at differentiere til definitiv endoderm herunder pankreatiske celletyper.
Det er tidligere rapporteret at neuronale celletyper, der er afledt fra ESC og som udtrykker nestin, kan give ophav til insulinproducerende celler. Disse cellers potentiale bliver her yderligere undersøgt, og vi kan vise at de nestin-positive celler ikke bliver til insulinproducerende celler. Det insulin man kan påvise i disse apoptotiske celler, skyldes at de optager insulin fra dyrkningsmediet. Det skyldes ikke insulin neosyntese i disse celler.
Nodal, som er medlem af TGF-β superfamilien, har vist sig at spille en afgørende rolle for dannelsen af endoderm i vertebrater. I dette arbejde har vi undersøgt betydningen af Nodal signalering ved in vitro differentiering af endoderm fra ESC. Resultaterne antyder, at Nodal signalering er vigtig for reguleringen af endoderm-specifikke gener, men det er ikke muligt i denne analyse at skelne mellem udviklingen af visceral og definitiv endoderm.
Når ESC sprøjtes ind i vævskompatible eller immunodefektive mus kan de give ophav til teratom, der er en form for godartet tumor. Vi undersøger her potentialet for teratomdannelse som en model for in vivo differentiering af pankreatiske celler. De indledende analyser af disse teratomer fra ESC indikerer, at der dannes tarm-lignende endoderm epitel, og det kan desuden delvis bekræftes, at differentiering af pankreatiske celletyper finder sted.