Analysis of congenital heart defects in Down Syndrome using induced pluripotent stem cells

A PhD project for the 2022 doctoral clinical fellows programme with Victor Tybulewicz (primary supervisor, Crick), Andreia Bernardo (Imperial College London) and Kuberan Pushparajah (King’s College London/St Thomas' Hospital).

Project description

Andreia Bernardo

Honorary Senior Research Fellow, National Heart and Lung Institute, Imperial College London

Kuberan Pushparajah

Clinical Senior Lecturer in Paediatric Cardiology, School of Biomedical Engineering & Imaging Sciences, King’s College London

Down Syndrome (DS), trisomy of human chromosome 21 (Hsa21), is characterised by a broad spectrum of phenotypes, including learning and memory deficits, craniofacial changes, early-onset Alzheimer’s disease and congenital heart defects (CHD) [1].

Each of these phenotypes arises from an extra copy of one or more of the ~230 genes on Hsa21. However, the identity of these causative genes is unknown as are the pathological mechanisms [2]. Understanding the genetic and mechanistic basis of these phenotypes is critical for the development of rational therapies, which are largely non-existent for this common human condition.

The CHD in DS typically include ventricular and atrioventricular septal defects and outflow tract defects. Using mouse models of DS, we have found that the mice show CHD similar to those seen in humans with DS and discovered that developing mutant hearts have reduced cell proliferation and mitochondrial dysfunction [3]. Furthermore, we have found that the causative genes for the CHD lie in a 39-gene region of the mouse genome with orthology to Hsa21 and that there must be at least 2 such genes. We now propose to study CHD in human DS cells, using Induced Pluripotent Stem Cells (IPSCs), to establish pathological mechanisms within different cardiovascular lineages and identify causative genes. 

Aims

  1. Determine the pathological consequences of trisomy of Hsa21 on cardiomyocytes.
  2. Determine the pathological consequences of trisomy of Hsa21 on endocardial and epicardial cells.
  3. Identify the dosage-sensitive genes on Hsa21 that cause CHD and establish the mechanisms by which they act.

The project will use matched isogenic pairs of IPSC lines from euploid and DS individuals [4]. These cell lines are genetically identical, expect for the presence of a third copy of Hsa21 in the DS lines. The student will grow the IPSC lines, differentiate them into ventricular cardiomyocytes, endocardial and epicardial cells using established protocols and analyse if the DS genotype causes changes in relevant cellular parameters, e.g. rates of cell division, mitochondrial mass, membrane potential and function.

The student will use RNAseq and mass spectroscopy to study the changes in the transcriptome and the proteome caused by trisomy 21 in different cardiac cell types. This will be extended to studies of human embryonic DS hearts, obtained from the Human Developmental Biology Resource. To identify causative genes, we will use CRISPR/Cas9 gene editing in DS IPSCs to inactivate one out of three copies of individual candidate genes and test if this mutation rescues phenotypes. The choice of candidate genes will be based on ongoing gene mapping studies in the mouse models of DS. The student will carry out cellular and biochemical analysis to determine the molecular mechanisms by which increased dosage of candidate genes contributes to CHD.

The student will be jointly supervised by Victor Tybulewicz and Andreia Bernardo. The Tybulewicz lab has extensive experience in the genetics of DS and in studies of embryonic heart development; the Bernardo lab has expertise in the growth and differentiation of IPSCs into cardiac lineage cells and in IPSC CRISPR/Cas9 gene editing.

The partner institution for this project is Imperial College London.

References

  1. Antonarakis, S. E. (2017). Down syndrome and the complexity of genome dosage imbalance. Nat Rev Genet 18: 147-163. PubMed abstract
  2. Lana-Elola, E., Watson-Scales, S. D., Fisher, E. M. and Tybulewicz, V. L. (2011). Down syndrome: searching for the genetic culprits. Dis. Model. Mech. 4: 586-595. PubMed abstract Down Syndrome
  3. Lana-Elola, E., Watson-Scales, S., Slender, A., Gibbins, D., Martineau, A., Douglas, C., . . . Tybulewicz, V. (2016). Genetic dissection of Down syndrome-associated congenital heart defects using a new mouse mapping panel. Elife 5: 10.7554/eLife.11614. PubMed abstract Down Syndrome
  4. Murray, A., Letourneau, A., Canzonetta, C., Stathaki, E., Gimelli, S., Sloan-Bena, F., . . . Nizetic, D. (2015). Brief report: isogenic induced pluripotent stem cell lines from an adult with mosaic down syndrome model accelerated neuronal ageing and neurodegeneration. Stem Cells 33: 2077-2084. PubMed abstract