TALENs Facilitate Single-step Seamless SDF Correction of F508del CFTR in Airway Epithelial Submucosal Gland Cell-derived CF-iPSCs

Abstract

Cystic fibrosis (CF) is a recessive inherited disease associated with multiorgan damage that compromises epithelial and inflammatory cell function. Induced pluripotent stem cells (iPSCs) have significantly advanced the potential of developing a personalized cell-based therapy for diseases like CF by generating patient-specific stem cells that can be differentiated into cells that repair tissues damaged by disease pathology. The F508del mutation in airway epithelial cell-derived CF-iPSCs was corrected with small/short DNA fragments (SDFs) and sequence-specific TALENs. An allele-specific PCR, cyclic enrichment strategy gave ~100-fold enrichment of the corrected CF-iPSCs after six enrichment cycles that facilitated isolation of corrected clones. The seamless SDF-based gene modification strategy used to correct the CF-iPSCs resulted in pluripotent cells that, when differentiated into endoderm/airway-like epithelial cells showed wild-type (wt) airway epithelial cell cAMP-dependent Cl ion transport or showed the appropriate cell-type characteristics when differentiated along mesoderm/hematopoietic inflammatory cell lineage pathways.

Figure 1

Isolation of a corrected clone after TALEN/SDF-mediated correction of genomic F508del CFTR in CF1-iPS4 cells. (a) Enrichment for corrected CF1-iPS4 cells co-transfected with 2 × 10⁷ 491z-SDFs/cell and the CFTAL-B expression vectors (lane 21). These cells showed the highest proportion of wtCFTR containing cells by AS-PCR and were distributed equally into a new multiwell plate. (b) AS-PCR analysis with primer pair CF1B/CF7C (Supplementary Table S2) (wt, top and middle) or CF1B/CF8C (Supplementary Table S2) (F508del, bottom) was again used to identify those cells with the highest proportion of wtCFTR DNA at each individual enrichment step followed by equal distribution of the cells from the well with the highest proportion of wtCFTR DNA into a new multiwell plate, etc (lanes 9 to 16), (first step: A4, second step: A4, third step: A3, fourth step: B2, fifth step: C2, sixth step: C1, seventh step: B3, eighth step: C2). (a and b) Lanes 2 to 8 reflect a control PCR titration analysis, mixing genomic DNA from a non-CF-iPS (wtCFTR) cell line (SC2-iPS_rs3) with CF1-iPS4 (F508delCFTR/F508delCFTR) at varying percentages. (c) Limiting dilution was used to isolate single cell-derived clonal populations of corrected cells from population C2 of the sixth enrichment step (EC₆). AS-PCR analysis was performed with primer pairs CF1B/CF7C or CF1B/CF8C on DNA from single colonies following limited dilution. Banding patterns for the wtCFTR/wtCFTR, heterozygote (wtCFTR/F508delCFTR), and homozygous mutant (F508delCFTR/F508delCFTR) were reconstructed by mixing the genomic DNA of a non-CF-iPS cell line (SC2-iPS_rs3) with CF1-iPS4 (F508del) at varying percentages (lanes 2 to 7). Clone c1 shows a pattern consistent with that of the wtCFTR/F508delCFTR heterozygote (lanes 9 and 10), while clones 2 to 20 all show a F508delCFTR/F508delCFTR pattern (Supplementary Figure S3, Supplementary Table S2).

Figure 2

Characterization of the CF1-iPS4c1 corrected cell line. (a) DNA sequencing histograms of non–allele-specific PCR products using CF1 and CF5 primers (Supplementary Table S2) from CFTR exon 11 in CF1-iPS4c1. The arrow indicates where the wt and F508del alleles on each chromosome diverge. (b) AS RT-PCR of RNA isolated from the parental CF1-iPS4 and from the CF1-iPS4c1 cells. The primer pair CF17/CF7C is selective for wtCFTR expression and CF17/CF8C (Supplementary Table S2) indicates expression of F508delCFTR mRNA. (c) Antibody staining for expression of pluripotence markers (Supplementary Table S1) with the CF1-iPS4c1–corrected cells (P5.55.21). (d) CF1-iPS4c1 cultures generated directly from the embryoid bodies demonstrate the potential for the corrected cells to differentiate into three germ layers in vitro. Immunostaining indicated endoderm (α-fetoprotein), mesoderm (α-smooth muscle actin), and ectoderm (TUJ1 (Supplementary Table S1)). (e) Southern blot analysis of EcoRV- and XbaI-digested genomic DNA (gDNA) isolated from untransfected CF1-iPS4 (parental cells, PC) as well as the CF1-iPS4c1 and D2-corrected clones. The wtCFTR-SDF (491z-SDF) was used as a probe for random integration or the endogenous CFTR locus. (f) Summary of off-targeting event analysis. The first four columns list predicted off-target sites for TALEN pair CFTAL-B as described in Supplementary Table S3. Column 5 indicates the results of sequencing. (-) = Corrected clone, CF1iPS4c1, P5.55.29, has no mismatches and Indels (deletions and/or insertions) compared with the CF1-iPS4 P5.50, parental cell line; (ND) = not done due to sequence analysis failure; a = CF1-iPS4 and corrected clone have identical nucleotide polymorphisms when compared to hg19 (UCSC human genome 19).

Figure 3

Functional analysis of corrected CF1-iPS4c1 cells. (a) CFTR Cl transport and expression of ZO-1 in CF1-iPS4c1 cells grown to confluent cell sheets in the presence or absence of CFBE41o- cells. Cells were grown under ALI culture conditions on day 12 and later. (b) Transepithelial Cl current recordings across CF1-iPS4c1 cells alone (i), co-cultures of CF1iPS4c1/CFBE41o-cells (ii, iii), and CFBE41o-cell monolayers, (control, d11) (iv); Fsk, 20 μmol/l forskolin; inh172, 50 μmol/l CFTRinh₁₇₂. Corresponding immunostaining for tight junction formation (Supplementary Table S1) in CF1-iPS4c1 islands or CFBE41o- cells alone was indicated below. (c) Representative transepithelial Cl currents (I_Cl) in positive control FHTF-iPS1 cells (wt/wt), CF1-iPS4c1 cells (wt/F508del), or negative control CF1-iPS4A1 cells (F508del/F508del) grown in CFBE41o- co-culture for 13 days. Current deflections indicate 1 mV pulses applied every 60 seconds to monitor transepithelial resistance. Amiloride (20 µmol/l) was added to the apical surface to inhibit Na absorption, while forskolin (20 µmol/l) and VX-770 (5 µmol/l) were added to the serosal and apical surface, respectively, to stimulate CFTR Cl currents. CFTRinh₁₇₂ (50 µmol/l) was added to the apical surface to verify inhibition of functional CFTR. (d) The bar chart reflects the inhibition of CFTR Cl currents by CFTRinh₁₇₂ in CF1iPS4A1, CF1iPS4c1, and FHTFiPS1 grown in CFBE41o- co-culture for 13 and 15 days, and CFBE41o- cells. Cl current changes (▵I_Cl) were determined from transepithelial recordings of Cl currents (I_Cl) after standardizing the drift. Results represent the mean ± SE (n = 4; *P < 0.05 versus CF1-iPS4A1; ANOVA followed by Holm-Sidak test). ALI, air–liquid interface.

Figure 4

Directed airway differentiation and functional CFTR expression. (a) Time course of a defined airway differentiation protocol. (b, Supplementary Table S1) Immunostaining for FOXJ1 (red, ciliated cell marker) and NKX2.1 (green, airway cell marker) with the overlay at days 43 and 61. Increased co-expression (orange) from day 43 to day 61 indicates prominent proximal airway differentiation and the presence of ciliated cells. Surfactant protein C (distal airway and alveolar type II cell marker, green) was detected in a few cells at day 61. Nuclei are stained with Hoechst 33342 (blue). (c, Supplementary Table S1) Immunostaining of CFTR protein expression of confluent CF1-iPS4c1cells grown on Snapwell inserts (filter) on day 18 and day 62. Orthogonal XZ image at day 62 shows abundant CFTR expression at the luminal surface. (d) Corresponding transepithelial I_Cl measurement from days 18, 43, and 61. Ussing assay was performed with pure cultures of CF1-iPS4c1 grown on Snapwell inserts in presence of a serosal-to-mucosal Cl gradient (120 mmol/l: 0 or 60 mmol/l) and in presence of amiloride (Amilo, 20 mmol/l), forskolin (Fsk, 20 mmol/l), and VX-770 (3 mmol/l). Transepithelial I_Cl was detected in response to CFTR-inh 172 (50 mmol/l) and glibenclamide (1 mmol/l). Voltage pulses (1 mV) were applied every 60 seconds and are cut off. Details for inhibition of I_Cl by CFTR-inh172 and glibenclamide are indicated in cultures for days 18, 43, and 61. Acute inhibition demonstrates CFTR activity in the CFTR-corrected iPSCs. ii. Average changes in chloride current (▵I_Cl) in response to CFTR-inh172 and iii. Glibenclamide at increasing times of differentiation. Data were pooled from measurements that were done on two consecutive days (days 61 and 62). Number of experiments is indicated in parenthesis (n = 1–4).