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Development of a proof of principle model for the therapeutical use of induced Pluripotent Stem (iPS) cells

Final Report Summary - GOODCELLS (Development of a proof of principle model for the therapeutical use of induced Pluripotent Stem (iPS) cells.)

The overall goal of this proposal was to develop an efficient treatment for Cystic Fibrosis (CF), the main genetic cause of death in Caucasian children, by combining techniques of gene and cellular therapy (Fig. 1).

To this end, human and mouse induced Pluripotent Stem Cells (iPSCs) carrying the p.F508del mutation in the CFTR gene were produced from homozygous subjects. iPSC lines were generated by retroviral transduction of the c-Myc, Klf4, Oct4, and Sox2 transcription factors (Objective 1). The newly created lines have been maintained in continuous culture for up to 6 months showing expression of stemness markers such as Nanog, Oct-4, Sox2 and SSEA1 (Fig. 2) and a normal karyotype. Differentiation properties of both human and mouse iPSCs were demonstrated by embryoid bodies and teratoma formation, respectively.

The correction of the p.F508del allele of the CFTR gene in iPSCs (Objective 2) relied on TALEN-based homologous recombination mediated by a targeting vector based on piggyback technology. TALENs pairs and targeting vectors were nucleofected into CF-iPSCs. Candidate positive clones were identified by negative selection with Ganciclovir and positive selection with puromycin. Bona fide targeted clones containing a corrected CFTR allele were validated by PCR analysis and sequencing (Fig. 3).

Once, repaired iPSC lines were well characterized, a two-step in vitro differentiation protocol was established (Objective 3). It mimics the natural process of lung embryonic development, and is able to produce foregut endodermal cells reproducibly (Fig. 4).

Finally, we have started a series of cell transplantation experiments aimed to define the conditions required for lung regeneration (Objective 4). The results are very preliminary, but after intravenous injection, iPSCs expressing Infrared fluorescent protein (IRFP) have a clear tendency to home into the lungs (Fig. 5).
[NOTE: See figures in the attached document]