Targeted correction and restored function of the CFTR gene in cystic fibrosis induced pluripotent stem cells.
Bottom Line: Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC) lines.We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele versus the other.The corrected cystic fibrosis iPSCs, when induced to differentiate in vitro, expressed the corrected CFTR gene; importantly, CFTR correction resulted in restored expression of the mature CFTR glycoprotein and restoration of CFTR chloride channel function in iPSC-derived epithelial cells.
Affiliation: Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center, Houston, TX 77030, USA.Show MeSH
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Mentions: We next examined expression of corrected CFTR mRNA and protein under in vitro differentiation conditions previously demonstrated to generate anterior foregut endoderm and primordial lung progenitors from mouse ESC (Longmire et al., 2012) and human ESC/iPSC (Green et al., 2011). In brief, after induction of definitive endoderm, inhibition of BMP/TGF-β signaling with NOGGIN/SB431542 was employed to enrich for anterior foregut endoderm. Subsequent exposure to growth factors implicated in lung development and maturation (WNT3a/KGF/FGF10/BMP4/EGF and retinoic acid) was then used to induce expression of NKX2-1, the earliest marker of commitment of endoderm to either a lung or thyroid epithelial cell fate (Figure 3A). Employing this protocol, mutant CF iPSCs (two independent clones, 17 and 28), corrected CF iPSCs (three clones, 17-9-C1, 17-14-C1, and 17-16-C1, independently obtained from correction of mutant clone 17), and WA09 hESCs efficiently generated definitive endoderm, as evidenced by co-expression of CXCR4 and C-KIT in >90% of cells (Figure S3A) and upregulated expression of endodermal transcriptional regulators, FOXA2 and SOX17 mRNA, by qPCR (data not shown). Further endodermal differentiation, for a total of 19 days in this protocol, subsequently upregulated expression of NKX2-1, SOX9, TP63, FOXP2, and FOXA2, suggesting commitment of at least a sub-population of cells within the endodermal culture to a lung epithelial cell fate (Longmire et al., 2012), and demonstrated increased expression of the CFTR target gene (Figure 3B), as expected from differentiated endodermally derived epithelia. Immunostaining confirmed that a subpopulation of cells co-expressed NKX2-1 and FOXA2 in this directed differentiation protocol (Figure S3B). Importantly, CFTR mRNA expressed on day 19 from differentiated mutant CF iPSCs (clone 17) reflected expression of both mutant ΔI507 and ΔF508 CFTR alleles, whereas differentiation of corrected iPSCs (17-16-C1) revealed co-expression of corrected WT and mutant ΔF508 CFTR mRNAs (Figure S3C). These results indicate appropriately regulated gene expression of the corrected WT allele, in comparison to the mutant ΔF508 allele, in the corrected iPSC-derived cells.
Affiliation: Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center, Houston, TX 77030, USA.