Genetic tracing of pancreatic transdifferentiation.

Isabelle Houbracken, Luc Baeyens, Jan De Jonge, Pedro Luis Herrera, Harry Heimberg, Luc Bouwens

Onderzoeksoutput: Meeting abstract (Book)

Samenvatting

Introduction: There is currently much interest in finding stem cells in pancreas tissue in order to generate insulin-producing beta-cells. Beta-cell transfer can cure diabetes but this therapy is hampered by donor shortage. Baeyens et al. (2005) have demonstrated that acinar cells of the exocrine pancreas can transdifferentiate in culture into beta-cells. During the phase of dedifferentiation that precedes the transdifferentiation, the acinar cells loose their differentiation characteristics (e.g. amylase-expression). Therefore it is very hard to demonstrate transitional cells expressing phenotypical markers of the original acinar cells (amylase) and of the final beta cell phenotype (e.g. insulin). We aim to develop a method for genetic tracing by which the phenotype of the precursor cells can unambiguously be demonstrated.

Material & Methods: To perform the tracing, two constructs, Amyp-Cre-Ires-ECFP and CMVp-Lox-RFP-Lox-Ires-EGFP, will be cloned in adeno- and lentiviral expression vectors. Acinar cells will be transduced by these vectors at the time of isolation. The amylase promoter will be actively producing Cre and ECFP in acinar cells, while all infected cells will be positive for RFP and EGFP. The Cre protein will remove the floxed RFP and the efficiency of double infection in acinar cells can be evaluated by counting the number of blue-green (not red) fluorescent cells. Then cells will be cultured under conditions that induce exocrine-to-endocrine transdifferentiation. Direct fluorescence combined with immunocytochemical staining for beta cell markers will allow to identify the transdifferentiated cells that became insulin+ and still are GFP+RFP-.

Results: The first phase of making the viral vectors is still going on.

Discussion & conclusion: No results can yet be discussed. If the transdifferentiation of acinar to beta-cells can be confirmed by this tracing method, differentiated exocrine tissue can be considered as an abundant source to generate beta-cells for clinical applications. Transdifferentiation or epigenetic cell re-programming can provide an interesting alternative to stem cell-based approaches.

Abbreviations: Amyp: amylase-specific promotor; Cre: Cre recombinase; Ires: internal ribosomal entry site; CFP, RFP and GFP: respectively cyan, red and green fluorescent protein; CMVp: cytomegalovirus ubiquitous promotor; Lox: target site for Cre.
Originele taal-2English
Titel9th Maastricht Medical Students Research Conference 2005
StatusPublished - 23 mrt 2005

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