Pneumologie 2015; 69 - A8
DOI: 10.1055/s-0035-1556600

Generation of disease-specific iPSCs and development of transgenic reporter cell lines for cystic fibrosis disease modelling and drug screening

M Schubert 1, 2, S Merkert 1, 2, A Haase 1, 2, L Engels 1, 2, R Haller 1, 2, N Lachmann 3, T Moritz 3, B Tümmler 4, L Galietta 5, U Martin 1, 2
  • 1Leibniz Research Laboratories for Biotechnology and Artificial Organs, Hannover Medical School
  • 2Biomedical Research in Endstage and Obstructive Lung Disease (BREATH), Member of the German Center for Lung Research (DZL)
  • 3Institute of Experimental Hematology, Hannover Medical School
  • 4Clinic for Paediatric Pneumology, Allergology and Neonatology, Hannover Medical School
  • 5Istituto Giannina Gaslini, Genova, Italy

Introduction:

Cystic fibrosis (CF) is a genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene coding for a cAMP-activated chloride-channel expressed in different epithelia. So far, immortalized cell lines overexpressing mutant CFTR-variants have been used to screen compound libraries. In fact, CFTR-protein-modulators have been identified, but show modest effects at best. Obviously, the complexity of the mutant CFTR-maturation and turnover kinetics including the influence of genetic modifiers require the use of advanced personalized cellular models that closely recapitulate the properties of the most clinically affected organs. To address these unmet needs we focus on the generation of induced pluripotent stem cell (iPSC) lines from CF-patients with the most common trafficking mutation (F508del), but with different phenotypes.

Methods:

CF-iPSCs were generated via reprogramming of CD34+ cells isolated from small volumes of non-mobilized peripheral blood. The resulting CF-iPSCs were analysed regarding their karyotype, pluripotency status and potential to differentiate. Moreover, different transgenic iPSC and embryonic stem cell (ESC) lines were generated overexpressing a halide sensitive yellow fluorescent protein (YFP) monitoring CFTR-function, in combination with the overexpression of an artificial CFTR or an endogenous CFTR-tomato-fluorescence-reporter.

Results:

Several CF-iPSC lines were established and characterized in detail. The generated YFP-reporter cell lines showed stable transgene expression also during in vitro differentiation and the general functionality of the YFP-reporter could be confirmed. Functional measurements to test the CFTR dependent halide sensitivity of our transgenic cell lines are currently ongoing and show promising results. Differentiation of YFP-expressing CFTR-tomato-reporter iPSCs towards cholangiocytes revealed YFP+/tomato+ cells.

Conclusions:

Hence, the stable integration of the halide reporter into CF-patient-specific iPSCs in combination with integration of the CFTR-tomato-reporter should enable disease modelling of F508del-based CF with regard to the individual genetic context and the implementation of high-throughput screening for novel correctors and potentiators of CFTR-trafficking mutations.

*Presenting author