A novel system for perfusion and real-time analysis of single hESC-derived cardiomyocytes

Lead Research Organisation: University of Nottingham
Department Name: School of Life Sciences

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

Publications

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Anderson D (2007) Transgenic enrichment of cardiomyocytes from human embryonic stem cells. in Molecular therapy : the journal of the American Society of Gene Therapy

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Braam SR (2010) Genetic manipulation of human embryonic stem cells in serum and feeder-free media. in Methods in molecular biology (Clifton, N.J.)

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Celiz AD (2014) Materials for stem cell factories of the future. in Nature materials

 
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 10/2013 
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 05/2015 
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 04/2014 
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 09/2014 
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 10/2013 
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 Charity/Non Profit
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 Charity/Non Profit
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