Modulation of transcription factor expression induces adult human pancreatic exocrine-endocrine transdifferentiation and protects rat beta-cells against cytokine-induced apoptosis

Onderzoeksoutput: PhD Thesis

Samenvatting

Given its recent clinical success, interest in treating insulin-dependent diabetes by transplantation of human insulin-producing beta-cells has been renewed. However, the severe limitation of the available amount of donor islet tissue only allows treatment of a very small fraction of patients. Because mature differentiated beta-cells cannot be expanded significantly in vitro, the need for innovative methods to establish an abundant supply of transplantable, insulin-producing tissue represents a highly relevant international research topic. A potential solution is to generate novel insulin-producing cells via differentiation of embryonic or adult stem cells or via transdifferentiation of mature cells. Although so far no conclusive evidence regarding the location of pancreatic cells with endocrine (trans)differentiation potential has been obtained, nor of the underlying mechanisms controlling their (trans)differentiation, indications for an endocrine (trans)differentiation potential in pancreatic duct cells urged us to examine their potential as a source for new beta-cells. Given the general idea that cell-specific transcription factors regulating the mature, differentiated phenotype of a particular cell are often also required for its embryonic development, we performed a direct comparison of the transcription factor expression profile of isolated adult human endocrine cells versus duct cells. The absence of an endocrine-specific transcription factor from the duct cell population would suggest its importance as an essential molecular endocrine developmental switch and, therefore, its usefulness as a target for experimental manipulation. In view of the fact that; (i) Foxa2, Pdx1/Ipf1 and Hnf6 are present in islet and duct cells, (ii) neurogenin-3 (ngn3) functions transiently immediately downstream from these factors during embryonic development of the mouse endocrine pancreas, and (iii) ngn3 is expressed at very low levels in duct and islet cells, we focused on the basic Helix-Loop-Helix (bHLH)-factor ngn3. We hypothesized that its low expression level in duct cells might constitute a physiological block to initiate an endocrine developmental program. Therefore, we designed ngn3 gain-of-function experiments, based on an adenoviral transduction system. Our work demonstrates the capacity for induction of an endocrine transdifferentiation program in isolated adult human duct cells. Adenoviral-mediated expression of ngn3 in adult duct cells recapitulates the embryonic pancreatic endocrine developmental program, culminating in the induction of insulin expression in a significant fraction of transduced duct cells. During this process, a hierarchical induction of the transcription factors Pax4, NeuroD1, Nkx6.1 and Pax6 - all known to be acting downstream from ngn3 during embryonic islet formation - is observed. Furthermore, a duct-to-beta-cell phenotypic switch is evident by the induction of endocrine and beta-cell specific markers (i.e. synaptophysin, chromogranin A, prohormone convertase 1/3, glucokinase and insulin). However, from a theoretical therapeutic standpoint, both the extent and yield of the endocrine transdifferentation process towards fully mature, glucose-sensitive insulin-producing cells needs to be greatly improved. To our knowledge, this is the first study demonstrating endocrine transdifferentiation of adult human duct cells induced by a single gene transfer. Furthermore, we show unambiguously that ngn3 is the switch for induction of a transcription factor-dependent program, involved in endocrine pancreas differentiation. Causative sequential activation of the factors downstream from ngn3 had never been dem
Originele taal-2English
Toekennende instantie
  • Vrije Universiteit Brussel
Begeleider(s)/adviseur
  • Bouwens, Luc, Co-Promotor
  • Heimberg, Henry, Promotor
Plaats van publicatieBrussels
StatusPublished - 2003

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