MODELLING CONGENITAL HEART DISEASES BY TBX1 KNOCKOUT IN A CARDIAC ORGANOID

Abstract

Congenital heart diseases (CHDs) have the highest incidence and mortality rate among birth defects worldwide. They arise from abnormal heart development, majority of which are associated with genetic variations present at birth. Tetralogy of Fallot (TOF) is one of the most common cyanotic CHD subtypes, and it is among the typical CHD phenotypes of patients with 22q11.2 Deletion Syndrome (22q11.2DS). TBX1 has been identified as a causal gene for TOF and other CHDs seen in 22q11.2DS. TBX1 is a key cardiac regulator during early heart development, and the abnormal expression of TBX1 has been proven to disrupt heart development, particularly the septation and elongation of the outflow tract (OFT). Therefore, TBX1 knockout (KO) was generated in this study to model CHDs with 3D cardiac organoids, a novel method that can represent early heart formation better than monolayer cardiomyocytes (CM). Following the protocol, human embryonic stem cell (hESC)-derived CMs were achieved, with positive staining of the CM marker, cTnT, at Day 15 of differentiation. The 3D cardiac organoids were successfully composed and maintained for six weeks, whereby consistently increased organoid sizes and regular beating activities were observed. Markers for different cardiac cell lineages other than CMs were observed, indicating the increased cellular complexity in cardiac organoids compared to the monolayer CMs. With the CRISPR/Cas9 ribonucleoprotein (RNP) method, a partial TBX1 KO (pKO) cell population was successfully generated. The H1 cells with TBX1 pKO displayed comparable cell morphology to the wildtype (WT) cells but delayed cell differentiation progress into CMs. On Day 8 of CM differentiation, markers TNNT2, NKX2-5, and ISL1, essential for differentiation, were decreased, indicating the delayed differentiation of the TBX1 pKO CMs. In conclusion, the deletion of TBX1 alters the in vitro hESC- CM differentiation, potentially supporting the notion of modelling complex CHDs with cardiac organoids in the future.