Generation of Isogenic iPS Cells by CRISPR/Cas Technology to Track TBX5 upon Cardiac Differentiation in a Patient-Specific iPS Model for the Holt–Oram Syndrome

Objectives: The Holt–Oram Syndrome (HOS) is characterized by upper limb malformation and congenital heart defects (CHD). HOS is inherited in an autosomal dominant manner and can be attributed to mutations in the TBX5 gene in more than 75% of the cases. So far, the relevance of TBX5 for heart morphogenesis and the HOS phenotype was investigated in animal models. We generated a human HOS patient-specific induced pluripotent stem cell (iPS) model using isogenic and Flag-tagged iPS lines to investigate the role of TBX5 in human cardiogenesis and identify dysregulations, associated with the development of the HOS.

Methods: iPS cells were generated from a HOS patient with a de novo mutation in the TBX5 gene and a severe clinical phenotype. CRISPR/Cas9 technology was used to correct the TBX5 mutation by nonhomologous recombination. Since existing anti-TBX5 antibodies show insufficiencies for different applications, a flag-tag was integrated into the TBX5 locus by homologous recombination (HR) to allow the specific detection of the TBX5 protein. To analyze TBX5 expression in cardiogenesis, iPS cells were directly differentiated to cardiomyocytes and gene expression was evaluated. The specific detection of TBX5 by antiflag antibodies was performed by flow cytometry, immunocytochemistry (ICC), and western blot (WB) analyses.

Results: Genome editing of the TBX5 locus by CRISPR/Cas9 in the patient-specific parental iPS line (TBX5±) yielded the corresponding isogenic iPS line (TBX5+/+). TBX5 gene expression upon cardiac differentiation was significantly reduced in the patient-specific parental iPS line (TBX5±). Correction of the TBX5 mutation in the iPS line (TBX5+/+), turned TBX5 expression back to reference levels. Insertion of the flag-tag showed highly efficient HR yielding 16 heterozygous TBX5-Flag ± iPS clones and two homozygous TBX5-Flag+/+ iPS clones. Flow cytometry, ICC, and WB analyses confirmed specific expression of TBX5 protein via the flag-tag upon cardiac differentiation, with no signals for undifferentiated iPS cells, known to not express TBX5.

Conclusion: The human iPS model for HOS is well suitable to define a temporary expression pattern of TBX5 during cardiogenesis and to identify dysregulations possibly contributing to the development of HOS. The corrected TBX5+/+ iPS cells and the flag-tagged iPS lines represent an ideal in vitro system to carry out advanced comparative molecular analyses of TBX5, which previously have not been possible.

No conflict of interest has been declared by the author(s).