Dr Richard P. Davis: Genome editing and cardiac differentiation of pluripotent stem cells

Head of the Group: Dr Richard P. Davis


Research Scientists: Viola A. Tabel, Karina de Oliveira Brandao, Catarina Martins Grandela

Research technician: Mervyn Mol

 

Main Research Themes:

  • Investigate the genetic contribution to cardiac disorders using human PSCs.
  • Develop high-throughput techniques to perform targeted genetic modification of human PSCs and their derivatives.
  • Develop genetic reporters to study cardiac lineage specification and for functional assessment.

 

My lab uses human pluripotent stem cells (hPSCs) to investigate the genetic contribution to arrhythmic disorders of the heart. It is currently estimated that congenital cardiac channelopathies (inherited diseases that disturb the function of ion channels in the heart), affect between 1:2000–1:3000 individuals in the general population. Even more common are acquired arrhythmias, predominantly caused by an adverse response to medication, and are a major challenge to clinicians and pharmaceutical companies. These too have a significant heritable component.

Although hundreds of mutations have been associated with both forms of arrhythmia, two outstanding issues remain. Firstly, it is difficult to prove the identified mutation is causal, and secondly large differences in disease severity are seen even among patients with the same primary mutation. This variability in severity is related not only to the mutation type and position of the mutation within the gene, but can also be influenced by variants in regulatory regions or in secondary modifier genes. It is likely that genomic loci identified in genome wide association studies (GWAS) contribute to this variable phenotype. However an inherent limitation of GWAS is that it illuminates regions in the genome associated with a trait, but does not identify the exact genetic variant and gene mediating that effect.

We have demonstrated that hPSCs, namely embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) generated by reprogramming somatic cells, serve as suitable in vitro disease models. These cells can generate the major cardiomyocyte subtypes (atrial, ventricular and pacemaker cells; Figure 1), and as many cardiac diseases are autonomous to the cardiomyocyte, hPSCs are promising in vitro paradigms for understanding disease pathophysiology and for identifying pathways to target in ameliorating the conditions.

We have also developed very efficient pipelines to genetically modify hPSCs, using either conventional gene targeting protocols or through novel nuclease-based approaches. This means we can either correct or introduce single nucleotide mutations in either patient hiPSCs or control hPSC lines to understand the contribution of that mutation to the disease. Additionally we have shown that we can use this same technology to target reporter genes, such as GFP or RFP, to key cardiac-specific genes (Movie 1). This allows us to not only study cardiac lineage development, but also selectively isolate particular cardiomyocyte subpopulations. These isolated cardiomyocytes then can be used to investigate the functional consequences of the variant on the ion channel, at both the electrophysiological and molecular levels.

However current methods (manual patch clamp electrophysiology) to assess the consequence of a genetic variant on the physiological properties of the cardiomyocyte are labour-intensive and time-consuming. Optogenetics, the amalgamation of optics with genetic engineering for precise monitoring of cellular processes, has found numerous applications within neurobiology but the possibilities in cardiac research are only now emerging. Using such an approach with hPSC-derived cardiomyocytes could offer a scalable strategy to identify and functionally validate the causal genes and the genetic variants contributing to arrhythmic disorders.

Therefore the key goals of my group are to:

1) Establish a scalable platform to measure a cardiomyocyte’s electrical activity through imaging and;

2) Develop novel gene-editing tools this will provide us with a medium throughput approach to functionally assess the contribution of disease-specific mutations as well as GWAS-linked variants to arrhythmias.

 

Grants:

  • ERC Starter’s Grant – STEMCARDIORISK
  • NWO VIDI fellowship - ILLUMINATE

 

Key Publications:

van den Berg CW, Elliott DA, Braam SR, Mummery CL, Davis RP. Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes Under Defined Conditions.Methods Mol Biol. 2016;1353:163-80.

van den Berg CW, Okawa S, Chuva de Sousa Lopes SM, van Iperen L, Passier R, Braam SR, Tertoolen LG, del Sol A, Davis RP, Mummery CL. Transcriptome of human foetal heart compared with cardiomyocytes from pluripotent stem cells. Development. 2015; 142:3231-8.

Bellin M, Casini S, Davis RP, D'Aniello C, Haas J, Ward-van Oostwaard D, Tertoolen LG, Jung CB, Elliott DA, Welling A, Laugwitz KL, Moretti A, Mummery CL. Isogenic human pluripotent stem cell pairs reveal the role of a KCNH2 mutation in long-QT syndrome. EMBO J. 2013 Dec 11;32(24):3161-75.

Davis RP, Casini S, van den Berg CW, Hoekstra M, Remme CA, Dambrot C, Salvatori D, Oostwaard DW, Wilde AA, Bezzina CR, Verkerk AO, Freund C, Mummery CL. Cardiomyocytes derived from pluripotent stem cells recapitulate electrophysiological characteristics of an overlap syndrome of cardiac sodium channel disease. Circulation. 2012 Jun 26;125(25):3079-91.

Davis RP, van den Berg CW, Casini S, Braam SR, Mummery CL. Pluripotent stem cell models of cardiac disease and their implication for drug discovery and development. Trends Mol Med. 2011 Sep;17(9):475-84.

Davis RP, Grandela C, Sourris K, Hatzistavrou T, Dottori M, Elefanty AG, Stanley EG, Costa M. Generation of human embryonic stem cell reporter knock-in lines by homologous recombination. Curr Protoc Stem Cell Biol. 2009 Nov;Chapter 5:Unit 5B.1 1.1-34.

Davis RP, Costa M, Grandela C, Holland AM, Hatzistavrou T, Micallef SJ, Li X, Goulburn AL, Azzola L, Elefanty AG, Stanley EG. A protocol for removal of antibiotic resistance cassettes from human embryonic stem cells genetically modified by homologous recombination or transgenesis. Nat Protoc. 2008;3(10):1550-8.

 

Full Publication list can be found here.