Induced pluripotent stem cell modelling of HLHS underlines the contribution of dysfunctional NOTCH signalling to impaired cardiogenesis

Abstract

Hypoplastic left heart syndrome (HLHS) is among the most severe forms of congenital heart disease. Although the consensus view is that reduced flow through the left heart during development is a key factor in the development of the condition, the molecular mechanisms leading to hypoplasia of left heart structures are unknown. We have generated induced pluripotent stem cells (iPSC) from five HLHS patients and two unaffected controls, differentiated these to cardiomyocytes and identified reproducible in vitro cellular and functional correlates of the HLHS phenotype. Our data indicate that HLHS-iPSC have a reduced ability to give rise to mesodermal, cardiac progenitors and mature cardiomyocytes and an enhanced ability to differentiate to smooth muscle cells. HLHS-iPSC-derived cardiomyocytes are characterised by a lower beating rate, disorganised sarcomeres and sarcoplasmic reticulum and a blunted response to isoprenaline. Whole exome sequencing of HLHS fibroblasts identified deleterious variants in NOTCH receptors and other genes involved in the NOTCH signalling pathway. Our data indicate that the expression of NOTCH receptors was significantly downregulated in HLHS-iPSC-derived cardiomyocytes alongside NOTCH target genes confirming downregulation of NOTCH signalling activity. Activation of NOTCH signalling via addition of Jagged peptide ligand during the differentiation of HLHS-iPSC restored their cardiomyocyte differentiation capacity and beating rate and suppressed the smooth muscle cell formation. Together, our data provide firm evidence for involvement of NOTCH signalling in HLHS pathogenesis, reveal novel genetic insights important for HLHS pathology and shed new insights into the role of this pathway during human cardiac development.

Whole Exome Sequencing Reveals the Major Genetic Contributors to Nonsyndromic Tetralogy of Fallot

“…Five out of seven variants tested were de novo, adding to the evidence for pathogenicity; the remaining variants were transmitted from unaffected parents indicating incomplete penetrance. Previous sequencing studies of CHD have identified an association of NOTCH1 variants in cardiac malformations including bicuspid aortic valve, aortic valve stenosis, coarctation of the aorta and hypoplastic left heart syndrome and TOF (43)(44)(45)(46)(47). However, the extent of NOTCH1 variant contribution to TOF has not been recognised until now.…”

Systematic Comparison of Retinal Organoid Differentiation from Human Pluripotent Stem Cells Reveals Stage Specific, Cell Line, and Methodological Differences

“…One hESC line (H9, WiCell) one neonatal iPSC line (Neo1, male [38,39]) and one adult iPSC line (AD3, from a 37-year-old female [40]) were adapted from MEF-supported to feeder-free culture conditions on 6 well plates coated with growth factor-reduced Matrigel basement membrane matrix (GFRM; Corning, New York) in TeSR1 (Stem Cell Technologies, Vancouver, Canada) defined medium. Cells were fed daily and passaged every 5 days at a ratio of up to 1:10 using 0.02% versene EDTA (Lonza, Basel, Switzerland).…”

Deleterious genetic variants inNOTCH1are a major contributor to the incidence of non-syndromic Tetralogy of Fallot

“…Previous sequencing studies of CHD have identified an association of NOTCH1 variants in left-sided cardiac malformations including bicuspid aortic valve, aortic valve stenosis, coarctation of the aorta and hypoplastic left heart syndrome (27)(28)(29)(30). Like TOF, the causative NOTCH1 variants described for these conditions are mostly missense with a small number of LOF variants located throughout the NOTCH1 protein.…”