A novel system for perfusion and real-time analysis of single hESC-derived cardiomyocytes
Lead Research Organisation:
University of Nottingham
Department Name: Sch of Biology
Abstract
About one week after fertilization between egg and sperm, the cells of the developing human embryo are primed to start forming all the cell types in the body. Scientists have shown that cells can be isolated at this early stage of development and then grown in the laboratory to produce large numbers of human embryonic stem cells (hESCs). Even in the laboratory, these cells can also be induced to make all cell types in the body. This may provide new opportunities to study early human development, understand more about genetic disorders or provide new sources of transplantable cells for conditions such as cardiovascular or Parkinsons disease. While hESCs could potentially have significant impact on our understanding of developmental biology and treatment of disease, the cell types derived from hESCs have not been studied in detail. For example, beating heart cells (cardiomyocytes) have been produced from hESCs but at best only limited information is available as to how they respond to different drugs or to the effect disruption of specific genes may have. Equally, no-one knows whether the characteristics of hESC-cardiomyocytes produced one day will be comparable to those produced the next. We have demonstrated that sufficient numbers of hESC-derived cardiomyocytes can be produced for detailed analysis. We now aim to engineer a new system that can simultaneously administer and analyze the effect of a variety of cardioactive drugs / gene modulatory agents. This will enable rapid development of hESC-cardiomyocytes as a tool for understanding human development and disease, as well as providing a platform to evaluate the utility of these cells for future medical application.
Technical Summary
Although many labs can successfully culture and spontaneously differentiate human embryonic stem cells (hESCs), the fundamental biology of undifferentiated and differentiated cells is poorly understood. To develop hESCs as scientific and therapeutic modalities, it will be vital to functionally characterize the differentiated cell types, preferably at the single cell level, to ensure reproducible preparations can be made. Thus, the aim of this proposal is to establish a platform technology to evaluate whether cohorts of single hESC-cardiomyocytes of comparable function can be produced both from different preparations using the same hESC line and from preparations using independently-derived lines Recently, we have standardized culture and cardiac differentiation between three independently-derived hESC lines, HUES-7, BG01 and NOTT1. We have now developed a high throughput differentiation protocol that utilizes growth factor induction to rapidly generate up to 45% spontaneously beating embryoid bodies (EBs). Beating areas can be readily disaggregated to single beating cardiomyocytes that are amenable to electrophysiology, calcium imaging and video edge detection, thus providing outputs to measure cardiomyocyte function and response In this proposal, we have designed and will engineer a semi-automated perfusion system that will interface with Multi Electrode Array, video edge and single cell fluorecence detection equipment to produce simultaneous real-time readouts for electrical and contractile activity, and fluorescence. This will facilitate rapid evaluation of functionality and reproducibility of different hESC-cardiomyocyte preparations as well as responsiveness to challenge with chronotropic agents. Finally, we will provide proof of principle that the system is amenable to delivery of lentiviral vectors designed for gene overexpression or knockdown in hESC-cardiomyocytes, thus establishing a novel route to studying gene function within these cells
Organisations
People |
ORCID iD |
Chris Denning (Principal Investigator) | |
Stephen Hill (Co-Investigator) |
Publications
Celiz AD
(2015)
Discovery of a Novel Polymer for Human Pluripotent Stem Cell Expansion and Multilineage Differentiation.
in Advanced materials (Deerfield Beach, Fla.)
Denning C
(2006)
Common culture conditions for maintenance and cardiomyocyte differentiation of the human embryonic stem cell lines, BG01 and HUES-7.
in The International journal of developmental biology
Denning C
(2016)
Cardiomyocytes from human pluripotent stem cells: From laboratory curiosity to industrial biomedical platform.
in Biochimica et biophysica acta
Denning C
(2008)
Cardiomyocytes from human embryonic stem cells as predictors of cardiotoxicity
in Drug Discovery Today: Therapeutic Strategies
Dick E
(2013)
Exon skipping and gene transfer restore dystrophin expression in hiPSC-cardiomyocytes harbouring DMD mutations.
in Stem cells and development
Dick E
(2013)
Exon skipping and gene transfer restore dystrophin expression in human induced pluripotent stem cells-cardiomyocytes harboring DMD mutations.
in Stem cells and development
Dick E
(2010)
Evaluating the utility of cardiomyocytes from human pluripotent stem cells for drug screening.
in Biochemical Society transactions
Dick E
(2011)
Faster generation of hiPSCs by coupling high-titer lentivirus and column-based positive selection.
in Nature protocols
Dick E
(2011)
Two new protocols to enhance the production and isolation of human induced pluripotent stem cell lines.
in Stem cell research
Dixon JE
(2016)
Highly efficient delivery of functional cargoes by the synergistic effect of GAG binding motifs and cell-penetrating peptides.
in Proceedings of the National Academy of Sciences of the United States of America
Description | The goal was to develop a novel perfusion system for analysing the response of human embryonic stem cell (hESC) derived cardiomyocytes after challenge with pharmacology and gene knockdown or overexpression. Although the limited time and personnel resources (no staff were funded) on this 18 month proposal restricted completion of some parts of the project, overall we made excellent progress, achieving 8 peer reviewed publications (including Nature Methods and Nature Protocols) and moved towards filing one patent. Thus, the outcomes contributed to UK science by improving scalable methods of hESC culture, differentiation and genetic modification. This enabled sufficient hESC-cardiomyocytes to be produced for proof of principle studies to show that these cells will likely be a valuable tool in large Pharma drug screening and toxicology campaigns. |
Exploitation Route | Commercial utility demonstrated by the follow grant awards, which are partly sponsored by industry: - BBSRC industrial partnership award with Syngenta. Grant no. BBG021821/1. Stem cell screening of human nutrient-gene interactions at the epigenetic level. 1.8.09-1.7.13. £1.3 million (Co-I) • SC4SM (Stem Cells for Safer Medicine Government / Private partnership Call). Developing hESC-derived cardiomyocytes as predictors of cardiotoxicity. 1.5.09-1.5.10. £80,000 (PI) • Bioprocessing Research Industry Club (BRIC) Grant BB/G010390/1. Developing scalable and standardised manufacturing methods for human PSCs. 1.1.09-31.11.12. £900,000 (Co-I) • Bioprocessing Research Industry Club (BRIC) Grant no. BB/G010285/1. Non-invasive biophotonics tool for phenotypic identification of pluripotent stem cells and their progeny. 1.3.09-1.3.12. £654,201 (Co-I) MEA detection of hESC-cardiomyocyte electrophysiology: We established the first MEA system in the UK for non-invasive, realtime analysis of the electrophysiological responses in hESC-cardiomyocytes. Improved hESC culture strategies. Ultimately, we demonstrated the MCT method could support 14 different hESC lines derived in 5 countries, even though the lines were originally cultured in the most diverse conditions available, varying in substrate, passaging method and medium composition (Braam et al., Nature Methods, 2008; Braam et al., Nature Protocols, 2008). Scalable hESC culture strategies, relevant to the automation industry (Thomas et al., Biotech & Bioeng 2009) Transgenic enrichment of hESC-cardiomyocytes: Published in Anderson et al., Molecular Therapy, 2007. Shows the potential of enriched cell populations in drug testing. Funding: This grant underpinned other successful awards including: - British Heart Foundation (A rationale approach to improving and scaling production of hESC-cardiomyocytes; PG/07/092/23722) - BBSRC funding (Engineering an in vitro living pump; BB/F020619/1). - BBSRC industrial partnership award with Syngenta. Grant no. BBG021821/1. Stem cell screening of human nutrient-gene interactions at the epigenetic level. 1.8.09-1.7.13. £1.3 million (Co-I) • SC4SM (Stem Cells for Safer Medicine Government / Private partnership Call). Developing hESC-derived cardiomyocytes as predictors of cardiotoxicity. 1.5.09-1.5.10. £80,000 (PI) • MRC Grant no. 20970. New in vitro models of DMD by induced pluripotency in patient biopsies and gene knockdown in hESCs. 1.1.09-31.9.12. £540,000 (PI) • Bioprocessing Research Industry Club (BRIC) Grant BB/G010390/1. Developing scalable and standardised manufacturing methods for human PSCs. 1.1.09-31.11.12. £900,000 (Co-I) • Bioprocessing Research Industry Club (BRIC) Grant no. BB/G010285/1. Non-invasive biophotonics tool for phenotypic identification of pluripotent stem cells and their progeny. 1.3.09-1.3.12. £654,201 (Co-I) |
Sectors | Healthcare |
Description | Masters course |
Geographic Reach | Multiple continents/international |
Policy Influence Type | Influenced training of practitioners or researchers |
Impact | masters levels training in stem cell technologies |
Description | Asha E-term fellowship |
Amount | £250,000 (GBP) |
Organisation | Engineering and Physical Sciences Research Council (EPSRC) |
Sector | Public |
Country | United Kingdom |
Start | 12/2013 |
End | 11/2015 |
Description | BHF Centre for Regen Med |
Amount | £2,500,000 (GBP) |
Funding ID | P47352/ |
Organisation | British Heart Foundation (BHF) |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 09/2013 |
End | 09/2017 |
Description | BHF MyoD grant |
Amount | £300,000 (GBP) |
Funding ID | PG/14/59/31000 |
Organisation | British Heart Foundation (BHF) |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 04/2015 |
End | 05/2018 |
Description | BHF programme 2014-17 |
Amount | £1,125,000 (GBP) |
Organisation | British Heart Foundation (BHF) |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 03/2014 |
End | 04/2017 |
Description | EPSRC equip - seahorse |
Amount | £115,000 (GBP) |
Organisation | Engineering and Physical Sciences Research Council (EPSRC) |
Sector | Public |
Country | United Kingdom |
Start | 08/2014 |
End | 03/2015 |
Description | MRC capital equipment |
Amount | £714,000 (GBP) |
Funding ID | MR/L012618/1 |
Organisation | Medical Research Council (MRC) |
Sector | Public |
Country | United Kingdom |
Start | 09/2013 |
End | 03/2014 |
Description | NC3Rs-CRACK-IT phase 1 |
Amount | £100,000 (GBP) |
Organisation | National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) |
Sector | Public |
Country | United Kingdom |
Start | 12/2013 |
End | 06/2014 |
Description | NC3Rs-CRACK-IT phase 2 |
Amount | £1,000,000 (GBP) |
Organisation | National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) |
Sector | Public |
Country | United Kingdom |
Start | 01/2015 |
End | 01/2018 |
Title | genome editing |
Description | genome editing techniques and cell lines |
Type Of Material | Cell line |
Year Produced | 2013 |
Provided To Others? | Yes |
Impact | developing UK and EU portfolio of research tools and assisting collaborations / grants |
Description | Observer & Guardian on Sunday 2014 |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | Description of research to National newspapers follow up enquiries and public engagement |
Year(s) Of Engagement Activity | 2014 |