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Background: Gene correction via homology directed repair (HDR) in patient-derived induced pluripotent stem (iPS) cells for regenerative medicine are becoming a more realistic approach to develop personalized and mutation-specific therapeutic strategies due to current developments in gene editing and iPSC technology. Cystic fibrosis (CF) is the most common inherited disease in the Caucasian population, caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Since CF causes significant multi-organ damage and with over 2,000 reported CFTR mutations, CF patients could be one prominent population benefiting from gene and cell therapies. When considering gene-editing techniques for clinical applications, seamless gene corrections of the responsible mutations, restoring native “wildtype” DNA sequence without remnants of drug selectable markers or unwanted DNA sequence changes, would be the most desirable approach.Result: The studies reported here describe the seamless correction of the W1282X CFTR mutation using CRISPR/Cas9 nickases (Cas9n) in iPS cells derived from a CF patient homozygous for the W1282X Class I CFTR mutation. In addition to the expected HDR vector replacement product, we discovered another class of HDR products resulting from vector insertion events that created partial duplications of the CFTR exon 23 region. These vector insertion events were removed via intrachromosomal homologous recombination (IHR) enhanced by double nicking with CRISPR/Cas9n which resulted in the seamless correction of CFTR exon 23 in CF-iPS cells.Conclusion: We show here the removal of the drug resistance cassette and generation of seamless gene corrected cell lines by two independent processes: by treatment with the PiggyBac (PB) transposase in vector replacements or by IHR between the tandemly duplicated CFTR gene sequences.

Effect of MET treatment on the expression of Foxa2 mRNA. MET stimulation enhances the expression of mFoxa2 mRNA expression in SK7 ES cells. b-actin was used as internal control (p = 0.031). Values are the mean ± S.E. Statistical significance was determined by Student’s t-test. *; p

Diagrams of the apparatus for MET stimulation and differentiation flow of ES cells on M15 feeder cells. (A) Diagram of MET treatment in vitro. MES: mild electrical stimulation. ms; millisecond, pps; pulses per second. (B) Differentiation flow of ES cells into Pdx1-expressing cells within the definitive endoderm on M15 feeder cells. Ect: ectoderm, ME: mesoendoderm, Mes: mesoderm, End: endoderm, Pdx1: Pdx1-expressing cells. (PDF 44 kb)

To examine the role of sialic acid in the respiratory tract, the influence of neuraminidase from Clostridium perfringens was investigated on contractions of isolated guinea-pig and rat trachea and on histamine release from guinea-pig lung tissue. Treatment with 2.0 units/ml of neuraminidase at 37 degrees C and pH 7.4 for 30 min caused an approximately 60% removal of total N-acetylneuraminic acid, a representative sialic acid, from muscle from guinea-pig and rat trachea. Neuraminidase concentration dependently induced histamine release from guinea-pig chopped lung tissue, but has no effect on contractions produced by acetylcholine, histamine and 5-hydroxytryptamine. Pretreatment with 2.0 units/ml of neuraminidase inhibited the contraction induced by antigen (ovalbumin) or compound 48/80. These findings suggest, at least in part, that sialic acids sensitive to neuraminidase are involved in the regulation of histamine release but not tracheal contraction.

The influences of extracellular Ca2+ and Mg2+ concentrations on the basal secretion of glycoconjugates from rabbit trachea in organ culture were examined. Over 80% of the 35S-labeled and [3H]glucosamine-labeled glycoconjugates secreted by the trachea were digested upon incubation with chondroitinase ABC. The basal secretion did not occur in the medium at 4 degrees C, indicating an energy-dependent process. The basal secretion at 37 degrees C of 35S-labeled glycoconjugates was prominently suppressed in Mg(2+)-free Tyrode solution but not in Ca(2+)-free Tyrode solution containing ethyleneglycol bis(2-aminoethylether)tetraacetic acid (EGTA). In contrast, the basal secretion of [3H]glucosamine-labeled glycoconjugates was not affected by the Mg2+ concentration in the medium. The results suggest that extracellular Mg2+ largely contributes to sulfation of glycoconjugates basally secreted from rabbit trachea.