Those raised in Newfoundland are all to familiar with the "Newfoundland Curse". Young people, often men, suddenly dropping dead of heart failure. The loss of loved ones in Newfoundland's tight-knit communities was devastating. The disease runs in families, who often feared that they or their loved ones would be affected. This disease is formally known as arrhythmogenic right ventricular cardiomyopathy (ARVC), which impacts the hearts of those affected. Ground-breaking research at Memorial University's Faculty of Medicine by Drs. Terry-Lynn Young, Kathy Hodgkinson, Sean Connors and Daryl Pullman identified the mutation and pioneered treatment to help extend the life of those affected. The mutation is in a protein known as Transmembrane Protein 43 (TMEM43).
Though they live many years longer than before, the lives of those with ARVC are still compromised. Implants and transplants are part of the life of an ARVC patient. Very little is known about TMEM43, the protein responsible for ARVC. In collaboration with the Sudden Cardiac Death Team at Memorial University, we are working to identify the role of TMEM43 in healthy people as well as how the mutant leads to ARVC. We are using CRISPR Cas-9 to delete TMEM43 in iPSCs, that will then be differentiated into cardiomyocytes. By comparing the activity of beating TMEM43 null cardiomyocytes to the wild-type control, we can learn more about why the mutation in TMEM43 leads to such a devastating condition.
Once we are able to better understand the role of TMEM43, we will use patient-derived iPSCs to investigate the nuances of ARVC. Though the mutation is the same in all patients, ARVC presents differently in different patients for unexplained reasons. Men are more adversely affected than women and some people live a full life with minor symptoms, while others may suddenly die at a very young age. Through the patient-derived cells, we will be able to better understand the nuances of this condition.
AD293 cells CRISPR'd to delete TMEM43, the gene responsible for ARVC-5
Calcium imaging, used to assess the electrical properties of our iPSC-derived cardiomyocytes.
All cells in the body are in constant communication with the cells in their surroundings. Cells can communicate with various direct and indirect signalling pathways. One way direct cell-cell communication is facilitated is by the Connexin family of proteins. Connexins form transmembrane hexameric hemichannels called connexons. These can communicate with the extracellular space or dock onto the Connexin hemichannels of other cells for direct cell-cell communication through what are known as gap junctions. There are many Connexin isoforms in humans. For example, Connexin43 (Cx43), as shown with our CRISPR-Cas9 knock-out iPSCs.
We are interested in the role that Connexins play in the tissue specification that occurs during early embryonic development. Our current research focus is the early differentiation of stem cells into what are known as the three germ layers: endoderm, mesoderm and ectoderm. Each of these layers is responsible for forming specific organs and tissue of the body. For example, the ectoderm forms the nervous system, while the mesoderm forms tissues such as muscle and bone. Communication between cells during this differentiation is essential. Improper communication can result in too much or too little of a given germ layer, which can be harmful or even fatal.
We use several tools to probe the formation of the germ layers. In order to investigate the significance of communication facilitated by Cx43, we use our CRISPR'd Cx43 null iPSCs. By differentiating these cells into the various germ lines, we can learn the importance of Cx43 during the early stages of development. Another tool we use is a Cx43-GFP iPSC cell line. The GFP, or Green Fluorescent Protein, allows us to investigate where Cx43 is in the cell by using live-cell confocal microscopy.
In the future, we can see the downstream effects of Cx43 perturbation as tissues further specify from the three germ layers.
Immunocytochemistry of iPSCs with Cx43 deletion using CRISPR-Cas9.
Free-form embroid bodies, which are spheres of cells that naturally form the three germ layers. We can use these emboid bodies to investigate changes in germ layer development in Cx43 null cells.
Early Brain Development
Pannexins are a family of proteins that form hexameric channels that allow the release of small molecules such as ATP and calcium ions from the plasma membrane or endoplasmic reticulum.
Pannexin1 (Panx1) is highly expressed in brain tissue so we are currently investigating the role that Panx1 has in early brain development. Using the gene editing tool CRISPR-Cas9, we have generated a Panx1 knock-out cell line of human induced pluripotent stem cells (iPSCs). These cells are a remarkable tool to study development as they can replicate in vitro indefinitely and become any cell type in the body. Human models of human development and disease including human cerebral organoids may identify mechanisms that other models miss.
The tools we use to study early brain development are iPSC-derived brain organoids and monolayer cultures. Brain organoids are 3D, in vitro structures that capture many of the cell types and cytoarchitectures found in the developing brain. Most reminiscent of the cortex, these organoids can provide insight into Panx1's structure-function relationships during human brain development. The second tool, iPSC derived monolayer cultures of brain cells such as neural precursor cells, astrocytes and neurons, are used to investigate the molecular mechanisms of Panx1.
We can investigate the role of Panx1 through the generation of knock-out organoids and monolayer cultures from our Panx1 null iPSC lines. Further, we use pharmacological inhibition of Panx1 to investigate how the role of Panx1 changes temporally.
A ten day old cerebral organoid, immunolabeled for nuclei (blue), Pannexin1 (green), Nestin (red) and Sox2 (white).
A 40 day old cerebral organoids (brightfield image).