{
  "abstract": "Introduction Congenital Heart Disease (CHD) is the most common birth defect globally, with Tetralogy of Fallot (ToF) being a frequent manifestation in 22q11.2 Deletion Syndrome (DiGeorge Syndrome). The transcription factor TBX1 has been identified as the major driver of these defects. TBX1 is critically required for the development of the Second Heart Field (SHF), which contributes to the right ventricle and the outflow tract. However, the precise dosage-sensitive mechanisms of TBX1 in early human cardiac lineage specification remain to be fully elucidated. We aim to generate a TBX1-deficient human embryonic stem cell (hESC) model to investigate these mechanisms and ultimately facilitate the generation of Cardiac Organoids (COs) for modelling CHD.Methods To model TBX1 haploinsufficiency, we utilized CRISPR/Cas9 gene editing to target the conserved T-box DNA-binding domain (Exons 3–6) in hESCs (H9 cell line). Clonal cell lines were established and screened via PCR, with specific deletions characterized by Sanger sequencing. The impact of the deletion on TBX1 expression was quantified using qPCR and Western Blotting. Cellular phenotype was assessed by quantifying proliferation rates using the MTT assay. Cell proliferation rates were assessed using the MTT assay. Pluripotency status was evaluated by measuring the expression of core markers OCT4 and SOX2.Results We successfully identified clonal hESC lines with a heterozygous deletion of the TBX1 T-box domain. These clones exhibited a significant reduction (60%-80%, p<0.05) in TBX1 expression at both mRNA and protein levels. Phenotypically, while the TBX1-deficient clones maintained the expression of the pluripotency marker OCT4, they displayed a statistically significant reduction in cell proliferation rates compared to wild-type controls. Additionally, gene expression analysis revealed a significant upregulation of SOX2 in the TBX1-deficient clones.Conclusion These findings demonstrate that the integrity of the TBX1 T-box domain is essential for maintaining hESC proliferation. The observed upregulation of SOX2 suggests that TBX1 insufficiency may disrupt the balance of pluripotency factors, potentially biasing cells towards a different lineage rather the cardiac mesoderm. These established cell lines provide a robust platform for our ongoing work in generating 3D COs, which will allow us to further investigate how TBX1 dosage sensitivity disrupts heart development.",
  "authors": [
    {
      "affiliations": [
        "University of Manchester, Manchester, United Kingdom"
      ],
      "name": "Mingwei Li"
    },
    {
      "affiliations": [
        "University of Manchester, Manchester, United Kingdom"
      ],
      "name": "Yingjuan Liu"
    },
    {
      "affiliations": [
        "University of Manchester, Manchester, United Kingdom"
      ],
      "name": "David Talevera"
    },
    {
      "affiliations": [
        "University of Manchester, Manchester, United Kingdom"
      ],
      "name": "Sabu Abraham"
    },
    {
      "affiliations": [
        "University of Manchester, Manchester, United Kingdom"
      ],
      "name": "Bernard Keavney"
    }
  ],
  "title": "407 Establishing a human embryonic stem cell model of TBX1 deficiency to investigate the pathogenesis of congenital heart disease",
  "uid": "741feede-bd81-5362-8152-2325ec3bd5dd"
}