Generation of three CRISPR/Cas9 edited human induced pluripotent stem cell lines (DHMi005-A-5, DHMi005-A-6 and DHMi005-A-7) carrying a Holt-Oram Syndrome patient-specific TBX5 mutation with known cardiac phenotype and a FLAG-tag after exon 9 of the TBX5 gene.

TBX5 is a transcription factor (TF) playing essential role during cardiogenesis. It is well known that TF mutations possibly result in non- or additional binding of the DNA due to conformational changes of the protein. We introduced a Holt-Oram Syndrome (HOS) patient-specific TBX5 mutation c.920_C > A heterozygously in a healthy induced pluripotent stell cell (iPSC) line. This TBX5 mutation results in conformational changes of the protein and displayed ventricular septal defects in the patient itself. Additionally we introduced a FLAG-tag on the TBX5 mutation-carrying allele. The resulting heterozygous TBX5-FLAG iPSC lines are a powerful tool to investigate altered TF activity bonding.

Generation of two CRISPR/Cas edited human induced pluripotent stem cell lines (DHMi005-A-3 and DHMi005-A-4) carrying a FLAG-tag after exon 9 of the TBX5 gene.

Although TBX5 plays a major role during human cardiogenesis and initiates and controls limb development, many of its interactions with genomic DNA and the resulting biological consequences are not well known. Existing anti-TBX5-antibodies work very inefficiently in certain applications such as ChIP-Seq analysis. To circumvent this drawback, we introduced a FLAG-tag sequence into the TBX5 locus at the end of exon 9 prior to the stop codon by CRISPR/Cas9. The expressed TBX5-FLAG fusion protein can effectively be precipitated by anti-FLAG antibodies. Therefore, these gene-edited iPSC lines represent powerful cellular in vitro tools to unravel TBX5:DNA interactions in detail.

Establishment of an induced pluripotent stem cell line DHMi005-A from a healthy male proband.

We generated an induced pluripotent stem cell (iPSC) line from a healthy male 29-year-old proband. Adipose fibroblasts (AFs) were reprogrammed using Sendai virus. Generated iPSCs showed typical stem cell morphology. From passage 9 on, iPSCs were free of virus. Pluripotency in the iPSCs was verified and spontaneous differentiation showed expression of all three germ layers. Karyotyping indicated no anomalies for the generated iPSCs. Many patient-specific iPSCs are generated from subcutaneous fat fibroblasts obtained during surgical procedure. The described control iPSC line was generated equally and therefore serves as an ideal control for adipose-fibroblast-based patient-specific iPSC lines in disease modeling.

Establishment of a patient-specific induced pluripotent stem cell line DHMi004-A from a male Holt-Oram syndrome patient with verified TBX5 mutation.

The Holt-Oram syndrome (HOS) is a rare autosomal dominant disorder, mostly based on mutations in the TBX5 gene. Patients show malformation of at least one upper limb along with congenital heart defects. The established induced pluripotent stem cell (iPSC) line was generated from a patient displaying pronounced and typical features of HOS and carrying a single-nucleotide change c.920_C>A leading to an amino acid change from proline to threonine at amino acid position 85, which appeared de novo. Adipose fibroblasts from the patient were reprogrammed using Sendai virus. Pluripotency of the iPSCs was fully demonstrated.