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Targeted correction and restored function of the CFTR gene in cystic fibrosis induced pluripotent stem cells.

Crane AM, Kramer P, Bui JH, Chung WJ, Li XS, Gonzalez-Garay ML, Hawkins F, Liao W, Mora D, Choi S, Wang J, Sun HC, Paschon DE, Guschin DY, Gregory PD, Kotton DN, Holmes MC, Sorscher EJ, Davis BR - Stem Cell Reports (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center, Houston, TX 77030, USA.

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In Vitro Differentiation of Corrected CF WT/ΔF508 iPSCs(A) Outline of the defined, step-wise differentiation protocol used to generate anterior foregut cell fates.(B) Gene expression analysis of day 19 differentiated mutant (17 and 28), corrected CF iPSCs (17-9-C1, 17-14-C1, and 17-16-C1), and WA09 hESCs indicates upregulation of lung and pan-endodermal markers. Data (mean ± SD, three well replicates) from a representative experiment further characterized in Figures 3C and 3G. See also Figure S3E.(C) Western blot analysis of protein lysates from day 19 differentiation cultures probed with a CFTR-specific antibody. Detection of Calnexin demonstrated equal sample loading for differentiated iPSC/hESC samples. See also Figures S3D and S3F.(D) Representative short-circuit current (Isc) traces of epithelial monolayers differentiated from mutant (17) and corrected (17-16-C1) CF iPSC by Ussing chamber analysis. Cells were treated with either DMSO (0.03%) or VX809 (3 μM) for 24 hr. After establishment of Cl− gradient and the addition of amiloride, monolayers were treated with forskolin and genistein followed by the administration of CFTR inhibitor 172. The change in Isc (μA/cm2) for each perturbation is shown. Only corrected clone 17-16-C1 demonstrates the presence of CFTR channels in the cell membrane, as evidenced by activation of cAMP-dependent short-circuit currents. The addition of CF corrector VX809 to differentiated clone 17 sample partially restores CFTR-mediated chloride activity.(E) Aggregated data of short-circuit current measurements from an experiment with differentiated mutant (17), with or without CF corrector VX809, and corrected (17-16-C1) CF iPSC (three transwell replicates per sample, mean ± SD.(F) Aggregated data of short-circuit current measurements from an experiment with differentiated mutant (17) and corrected (17-16-C1), with or without CF corrector VX809, and WA09 hESC control (five transwell replicates per sample, mean ± SD).(G) Aggregated data of short-circuit current measurements from an experiment including all independent mutant (17 and 28) and corrected (17-9-C1, 17-14-C1, and 17-16-C1) differentiated CF iPSC clones (three to six transwell replicates per sample, mean ± SD).(H) Aggregated data of short-circuit current measurements; graphed is the maximum change in short-circuit current resulting from the addition of forskolin and genistein. Shown is the mean ± SE. The comparison shown is between two mutant CF clones (17 and 28; total of four independent differentiated experimental samples, three to six transwell replicates per sample) and three corrected CF clones (17-9-C1, 17-14-C1, and 17-16-C1; total of five independent differentiated experimental samples, three to six transwell replicates per sample). Results were clustered by clonal cell line and experiment number; statistical analysis was performed using a linear mixed-effect model by restricted maximum likelihood to account for correlated replicates within the same experiment.See also Figure S3.
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fig3: In Vitro Differentiation of Corrected CF WT/ΔF508 iPSCs(A) Outline of the defined, step-wise differentiation protocol used to generate anterior foregut cell fates.(B) Gene expression analysis of day 19 differentiated mutant (17 and 28), corrected CF iPSCs (17-9-C1, 17-14-C1, and 17-16-C1), and WA09 hESCs indicates upregulation of lung and pan-endodermal markers. Data (mean ± SD, three well replicates) from a representative experiment further characterized in Figures 3C and 3G. See also Figure S3E.(C) Western blot analysis of protein lysates from day 19 differentiation cultures probed with a CFTR-specific antibody. Detection of Calnexin demonstrated equal sample loading for differentiated iPSC/hESC samples. See also Figures S3D and S3F.(D) Representative short-circuit current (Isc) traces of epithelial monolayers differentiated from mutant (17) and corrected (17-16-C1) CF iPSC by Ussing chamber analysis. Cells were treated with either DMSO (0.03%) or VX809 (3 μM) for 24 hr. After establishment of Cl− gradient and the addition of amiloride, monolayers were treated with forskolin and genistein followed by the administration of CFTR inhibitor 172. The change in Isc (μA/cm2) for each perturbation is shown. Only corrected clone 17-16-C1 demonstrates the presence of CFTR channels in the cell membrane, as evidenced by activation of cAMP-dependent short-circuit currents. The addition of CF corrector VX809 to differentiated clone 17 sample partially restores CFTR-mediated chloride activity.(E) Aggregated data of short-circuit current measurements from an experiment with differentiated mutant (17), with or without CF corrector VX809, and corrected (17-16-C1) CF iPSC (three transwell replicates per sample, mean ± SD.(F) Aggregated data of short-circuit current measurements from an experiment with differentiated mutant (17) and corrected (17-16-C1), with or without CF corrector VX809, and WA09 hESC control (five transwell replicates per sample, mean ± SD).(G) Aggregated data of short-circuit current measurements from an experiment including all independent mutant (17 and 28) and corrected (17-9-C1, 17-14-C1, and 17-16-C1) differentiated CF iPSC clones (three to six transwell replicates per sample, mean ± SD).(H) Aggregated data of short-circuit current measurements; graphed is the maximum change in short-circuit current resulting from the addition of forskolin and genistein. Shown is the mean ± SE. The comparison shown is between two mutant CF clones (17 and 28; total of four independent differentiated experimental samples, three to six transwell replicates per sample) and three corrected CF clones (17-9-C1, 17-14-C1, and 17-16-C1; total of five independent differentiated experimental samples, three to six transwell replicates per sample). Results were clustered by clonal cell line and experiment number; statistical analysis was performed using a linear mixed-effect model by restricted maximum likelihood to account for correlated replicates within the same experiment.See also Figure S3.

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.


Targeted correction and restored function of the CFTR gene in cystic fibrosis induced pluripotent stem cells.

Crane AM, Kramer P, Bui JH, Chung WJ, Li XS, Gonzalez-Garay ML, Hawkins F, Liao W, Mora D, Choi S, Wang J, Sun HC, Paschon DE, Guschin DY, Gregory PD, Kotton DN, Holmes MC, Sorscher EJ, Davis BR - Stem Cell Reports (2015)

In Vitro Differentiation of Corrected CF WT/ΔF508 iPSCs(A) Outline of the defined, step-wise differentiation protocol used to generate anterior foregut cell fates.(B) Gene expression analysis of day 19 differentiated mutant (17 and 28), corrected CF iPSCs (17-9-C1, 17-14-C1, and 17-16-C1), and WA09 hESCs indicates upregulation of lung and pan-endodermal markers. Data (mean ± SD, three well replicates) from a representative experiment further characterized in Figures 3C and 3G. See also Figure S3E.(C) Western blot analysis of protein lysates from day 19 differentiation cultures probed with a CFTR-specific antibody. Detection of Calnexin demonstrated equal sample loading for differentiated iPSC/hESC samples. See also Figures S3D and S3F.(D) Representative short-circuit current (Isc) traces of epithelial monolayers differentiated from mutant (17) and corrected (17-16-C1) CF iPSC by Ussing chamber analysis. Cells were treated with either DMSO (0.03%) or VX809 (3 μM) for 24 hr. After establishment of Cl− gradient and the addition of amiloride, monolayers were treated with forskolin and genistein followed by the administration of CFTR inhibitor 172. The change in Isc (μA/cm2) for each perturbation is shown. Only corrected clone 17-16-C1 demonstrates the presence of CFTR channels in the cell membrane, as evidenced by activation of cAMP-dependent short-circuit currents. The addition of CF corrector VX809 to differentiated clone 17 sample partially restores CFTR-mediated chloride activity.(E) Aggregated data of short-circuit current measurements from an experiment with differentiated mutant (17), with or without CF corrector VX809, and corrected (17-16-C1) CF iPSC (three transwell replicates per sample, mean ± SD.(F) Aggregated data of short-circuit current measurements from an experiment with differentiated mutant (17) and corrected (17-16-C1), with or without CF corrector VX809, and WA09 hESC control (five transwell replicates per sample, mean ± SD).(G) Aggregated data of short-circuit current measurements from an experiment including all independent mutant (17 and 28) and corrected (17-9-C1, 17-14-C1, and 17-16-C1) differentiated CF iPSC clones (three to six transwell replicates per sample, mean ± SD).(H) Aggregated data of short-circuit current measurements; graphed is the maximum change in short-circuit current resulting from the addition of forskolin and genistein. Shown is the mean ± SE. The comparison shown is between two mutant CF clones (17 and 28; total of four independent differentiated experimental samples, three to six transwell replicates per sample) and three corrected CF clones (17-9-C1, 17-14-C1, and 17-16-C1; total of five independent differentiated experimental samples, three to six transwell replicates per sample). Results were clustered by clonal cell line and experiment number; statistical analysis was performed using a linear mixed-effect model by restricted maximum likelihood to account for correlated replicates within the same experiment.See also Figure S3.
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fig3: In Vitro Differentiation of Corrected CF WT/ΔF508 iPSCs(A) Outline of the defined, step-wise differentiation protocol used to generate anterior foregut cell fates.(B) Gene expression analysis of day 19 differentiated mutant (17 and 28), corrected CF iPSCs (17-9-C1, 17-14-C1, and 17-16-C1), and WA09 hESCs indicates upregulation of lung and pan-endodermal markers. Data (mean ± SD, three well replicates) from a representative experiment further characterized in Figures 3C and 3G. See also Figure S3E.(C) Western blot analysis of protein lysates from day 19 differentiation cultures probed with a CFTR-specific antibody. Detection of Calnexin demonstrated equal sample loading for differentiated iPSC/hESC samples. See also Figures S3D and S3F.(D) Representative short-circuit current (Isc) traces of epithelial monolayers differentiated from mutant (17) and corrected (17-16-C1) CF iPSC by Ussing chamber analysis. Cells were treated with either DMSO (0.03%) or VX809 (3 μM) for 24 hr. After establishment of Cl− gradient and the addition of amiloride, monolayers were treated with forskolin and genistein followed by the administration of CFTR inhibitor 172. The change in Isc (μA/cm2) for each perturbation is shown. Only corrected clone 17-16-C1 demonstrates the presence of CFTR channels in the cell membrane, as evidenced by activation of cAMP-dependent short-circuit currents. The addition of CF corrector VX809 to differentiated clone 17 sample partially restores CFTR-mediated chloride activity.(E) Aggregated data of short-circuit current measurements from an experiment with differentiated mutant (17), with or without CF corrector VX809, and corrected (17-16-C1) CF iPSC (three transwell replicates per sample, mean ± SD.(F) Aggregated data of short-circuit current measurements from an experiment with differentiated mutant (17) and corrected (17-16-C1), with or without CF corrector VX809, and WA09 hESC control (five transwell replicates per sample, mean ± SD).(G) Aggregated data of short-circuit current measurements from an experiment including all independent mutant (17 and 28) and corrected (17-9-C1, 17-14-C1, and 17-16-C1) differentiated CF iPSC clones (three to six transwell replicates per sample, mean ± SD).(H) Aggregated data of short-circuit current measurements; graphed is the maximum change in short-circuit current resulting from the addition of forskolin and genistein. Shown is the mean ± SE. The comparison shown is between two mutant CF clones (17 and 28; total of four independent differentiated experimental samples, three to six transwell replicates per sample) and three corrected CF clones (17-9-C1, 17-14-C1, and 17-16-C1; total of five independent differentiated experimental samples, three to six transwell replicates per sample). Results were clustered by clonal cell line and experiment number; statistical analysis was performed using a linear mixed-effect model by restricted maximum likelihood to account for correlated replicates within the same experiment.See also Figure S3.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center, Houston, TX 77030, USA.

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Related in: MedlinePlus