Mitochondrial function and transition from glycolysis to oxidative phosphorylation in differentiating induced pluripotent stem cells

Lead Research Organisation: Newcastle University
Department Name: Institute of Human Genetics

Abstract

Induced pluripotent stem cells (iPSC) can be derived from human tissues such as the skin and have two characteristic properties. Firstly, we can grow them indefinitely in the laboratory in either small or large scale and secondy, by use of appropriate methods, they seem to be able to differentiate into almost all of the cell types found in the adult body. At a first glance this makes them similar to human embryonic stem cells which have been hailed as an attractive resource for regenerative procedures that involve transplantation of specific differentiated cells into human patients. Embryonic stem cells are derived from individual Human embryos so any cells we produce form these cell lines are likely to be rejected by the body's immune system. iPSC should not suffer from this problem if the cells we make from them are transplanted back into the same individual from which they were made but there is a potentially greater advantage of iPSC namely the creation of 'disease in a dish' models. This relies upon making an iPSC line from a patient suffering from a genetic disease then differentiating that iPSC line to the type of cells affected by the disease so we can see what problems arise and how we might be able to fix them. All of this relies upon the hiPSC line being able to differentiate into cells that are functionally identical to those found in the body. Available evidence and our pilot data suggest that although they may produce cells that look similar to adult cells (such as those of the bone marrow or the nervous system), these may not work as effectively as the genuine article therefore in order to realize the promise of iPSC technology, we have to find out why they are defective and develop ways to fix these problems. Our pilot data suggest that problems with mitochondria, the energy suppliers of the cell, may be a significant reason why iPSC and their differentiated problem could be dysfunctional. Mitochondria exist as distinct structures within the cell and while some of their components are produced from their own indepdent genes, the majority of their structure and function is controlled by genes residing in the cells nucleus. The ways in which mitochondria function are quite different in various cell types; for example hESC don't use them to generate energy very much but differentiated cells derived from hESC do. The human cells (somatic cells) we reprogram to make hiPSC fall into the latter category but it seems that the reprogramming is incomplete and hiPSC may still try to control their mitochondria in similar ways to the original somatic cells. This is potentially bad for the survival of the hiPSC but even worse, they may not be able to regulate mitochondria properly on differentiation. In view of this our objectives are to compare the mitochondria of hiPSC and hESC and then differentiate them to three cell types with differing energy requirements (and therefore different mitochondrial regulation). We will compare the expression of genes involved in mitochondrial function, analyse their control mechanisms and attempt to explain why this might not work optimally. Once this understanding is gained, we will be able to develop means to solve these problems and produce hiPSC with fullly functional mitochondria although this in turn may allow us to improve other aspects of iPSC function that rely upon completion of reprogramming. The successful execution of this project will be enormously beneficial not only to ours and other groups investigating iPSC biology but it will also promote development of disease treatments using these cells which will provide significant social and economic impact.

Technical Summary

Our pilot data suggest that the mitochondria of induced pluripotent stem cells (iPSC), may not function in an analogous manner to those of human embryonic stem cells (hESC). There are similarities in terms of mitochondrial genome copy number and mitochondrial mass in both hiPSC and hESC but mitochondrial membrane potential and production of superoxide are significantly lower in iPSC. In addition, several nuclear encoded genes central to mitochondrial function, are mis-expressed compared to hESC both in the pluripotent cells and their differentiated progeny. Crucially, the extent to which mis-expression occurs varies between isogneic iPSC lines suggesting that the causes of these differences may be epigenetic and implies that the epigenetic reprogramming originally required to generate the iPSC from dermal fibroblasts may have been incomplete. If this is a general phenomenon with iPSC, they could be a poor source of cells for regenerative medicine or disease modelling so we plan to analyse mitochondrial parameters such as morphology, membrane potential and expression levels of genes associated with mitochondrial function across a range of hiPSC and hESC lines and somatic cell types we can produce from them. This point is important since hiPSC and hESC do not use the oxidative phosphorylation ATP generation system intrinsic to the mitochondria to the same extent as many somatic cells. It is therefore important to determine that hiPSC are capable of generating the numbers of mitochondria required by the somatic cell types they differentiate into and that these mitochondria function in the same way as we would expect of the progeny of hESC. We will analyse mitochondrial numbers and morpholgy using established histochemical staining protocols but the key steps of this proposal will be analyses of gene expression using microarrays and epigenetic architecture of nuclear encoded mitchondrial associated genes using chromatin immunoprecipitation.

Planned Impact

Since the first publication detailing the derivation of Human iPSC in 2007, nearly two thousand articles have appeared in the scientific literature describing new methods to generate these cells and the attempts to develop protocols for their differentiation into somatic cells with potential clinical uses. Many studies of this type were already in progress using embryonic stem cells but the enormous advantage of iPSC is that they are isogenic with the individual from whom they are derived. If however they do not behave in the same predictable manner as ESC, they may not be so useful either as a source of cells in regenerative medicine or as in vitro models of human diseases. This proposal aims to determine if mitochondria in iPSC behave in a similar fashion to those of ESC and to explain any differences in terms of incomplete epigenetic reprogramming or other factors that contribute to mitochondrial function. Moreover, if the dysfunction results from problems with epigenetic regulation of nuclear encoded genes associated with mitochondrial activity, we hope to be able to 'repair' this so the mitochondria behave in a more normal fashion. In view of this, the most immediate group to benefit from our research will be the large number of academic groups who are working with iPSC as we have described in the academic beneficiaries section however the potential benefits of this work extend beyond the boundaries of purely academic research. Clinical trials are beginning with embryonic stem cell derived somatic cells so it is highly probable that iPSC represent the next logical step in development of stem cell based therapies if current problems with their growth and differentiation parameters can be overcome. A recent article in Nature Biotechnology (Nat Biotech 2010, 28(6):535-536) further underlines considerable industrial interest in pluripotent stem cells as sources of clinically useful tissue thus the results of this project are highly likely to be beneficial to the future utility of iPSC in commercial regenerative medicine. Regenerative medicine is likely to have a highly significant economic impact upon nations with the scientific and financial capabilities needed to implement its development. To set this statement in context, the USA currently spends $175.8 billion annually for the treatment of diabetes and myocardial infarction, two conditions which are primary targets for stem cell based regenerative therapies. The United Kingdom is a leading contributor to Stem Cell related research and if the results of this project allow the development of clinically useful iPSC, and these can undergo successful translation into commercial cell therapy products, the economic benefits to the UK will be considerable. The social impact of such developments cannot be understated. If effective treatments for the two conditions mentioned above and others arise, even indirectly from the results of this project we will have contributed in an effective way to improving the health and quality of life of significant numbers of individuals both in the UK and in other countries.

Publications

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Armstrong Lyle (2013) Epigenetics

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Armstrong Lyle Generating Collections of Human iPSC and ESC Lines: Establishing Best Practices for Sharing Protocols and Cell Lines in International Society for Stem Cell Research 11th Meeting

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Atkinson SP (2013) Potential for pharmacological manipulation of human embryonic stem cells. in British journal of pharmacology

 
Description Human induced pluripotent stem cells (hiPSC) utility is limited by variations in their ability to undergo lineage specific differentiation. We have undertaken a transcriptional comparison of human embryonic stem cell lines (hESC) and hiPSC lines and have shown that hiPSC are inferior in their ability to undergo neuroectodermal differentiation. We have identified CHCHD2, whose expression is much lower in hiPSC when compared to hESC and can be used as a marker to predict the neuroectodermal differentiation potential of hiPSC. We provide evidence that the hiPSC variability with respect to CHCHD2 expression and differentiation potential is caused by clonal variation during the reprogramming process and that CHCHD2 primes neuroectodermal differentiation of hESC and hiPSC through regulating the activity of the TGFß signalling pathway. The potential utility of using CHCHD2 as a marker for assessing and comparing the hiPSC clonal and/or line differentiation potential provides a tool for large scale differentiation and hiPSC banking studies.
Exploitation Route The preliminary investigations that led to this project suggested possible problems with regulation of mitochondrial oxidative phosphorylation in induced pluripotent stem cells. The data we obtained from this investigation indicate that a specific gene, CHCHD2, seems to be involved in controlling differentiation of pluripotent stem cells and may also have functions in the mitochondria but most importantly, these findings enhance our knowledge of the mechanisms that maintain pluripotency. This contributes to the development of more effective ways to use pluripotent stem cells for investigations in developmental biology, drug discovery and potentially as cell sources for regenerative medicine
Sectors Education

Healthcare

Manufacturing

including Industrial Biotechology

Pharmaceuticals and Medical Biotechnology

 
Description The knowledge gained from this investigation has contributed in part to our ability to use induced pluripotent stem cells to model a range of diseases in vitro. In turn, this has contributed to the foundation of s spin out company (Newcells Biotech Ltd) in which the grant holder (Prof L Armstrong) and Newcastle University hold equity
First Year Of Impact 2015
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description Innovative Medicines Initiative - EBiSC
Amount £500,000 (GBP)
Funding ID IMI grant agreement No 115582 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 03/2014 
End 12/2017
 
Description Innovative Medicines Initiative - StemBANNC
Amount £1,800,000 (GBP)
Funding ID IMI grant agreement No 115439 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 09/2012 
End 09/2017
 
Description Integrated MRes/PhD studentship
Amount £100,000 (GBP)
Organisation National Health and Medical Research Council 
Sector Public
Country Australia
Start 09/2012 
End 09/2016
 
Description MRC confidence in concept:
Amount £61,997 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 05/2016 
End 05/2017
 
Description Marie-Curie training fellowship
Amount € 250,000 (EUR)
Organisation Marie Sklodowska-Curie Actions 
Sector Charity/Non Profit
Country Global
Start 03/2017 
End 02/2020
 
Description NC3R: CRACK-IT challenge 20: METABODERM;
Amount £800,000 (GBP)
Organisation National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) 
Sector Public
Country United Kingdom
Start 09/2016 
End 09/2019
 
Description NCR3R CrackiT: 3D hiPSC derived laminated retina model: £ 99,981.87, Coinvestigator
Amount £99,981 (GBP)
Funding ID NC/C016106/1 
Organisation National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) 
Sector Public
Country United Kingdom
Start 01/2017 
End 06/2017
 
Description responsive mode
Amount £170,000 (GBP)
Organisation Macular Society 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2014 
End 03/2017
 
Description responsive mode
Amount £30,000 (GBP)
Organisation Newcastle upon Tyne Hospitals NHS Charity 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2014 
End 01/2016
 
Description responsive mode
Amount £49,990 (GBP)
Organisation Newcastle upon Tyne Hospitals NHS Charity 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2014 
End 10/2016
 
Company Name Newcells Biotech 
Description Newcells Biotech develops stem cell technology that removes the need for embryonic stem cells, for use in modelling and drug discovery. 
Year Established 2015 
Impact the company is in the process of establishing business with several major pharmaceutical companies
Website http://www.newcellsbiotech.co.uk
 
Description Accelerating drug discovery 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Presentation to ISCF Medicines Manufacturing Workshop, 10th August 2017, Newcastle University, UK
Year(s) Of Engagement Activity 2017
 
Description Concluding data from the StemBANCC project 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Concluding data from the StemBANCC project
Year(s) Of Engagement Activity 2017
 
Description ISSCR poster "A novel role for MiR1305 in regulation of pluripotent-differentiation balance, cell cycle and apoptosis in human pluripotent stem cells, 24th June 2016, San Francisco, USA 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Third sector organisations
Results and Impact presentation of data to ISSCR
Year(s) Of Engagement Activity 2016
 
Description ISSCR poster "Improving our understanding of autosomal dominant retinitis pigmentosa using PRPF31 patient specific induced pluripotent stem cells" 24th June 2016, San Francisco, USA, 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Third sector organisations
Results and Impact presentation of data to ISSCR
Year(s) Of Engagement Activity 2016
 
Description ISSCR poster W2084, June 22nd 2016, San Francisco, USA, "CHCHD2 primes the differentiation potential of human induced pluripotent stem cells to neuroectodermal lineages" 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Third sector organisations
Results and Impact presentation of data to the International Society for Stem Cell Research
Year(s) Of Engagement Activity 2016
 
Description International Society for Stem Cell Research (ISSCR), Boston MA, USA. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Invited speaker in session "generating collections of human iPSC and ES lines". Possible collaborations explored

none yet
Year(s) Of Engagement Activity 2013
 
Description Manchester Tissue Regeneration & Stem Cell Network seminar series, Manchester University 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact described my groups work on induced pluripotent stem cells to academics in Manchester University. Generated useful and stimulating discussions

none yet
Year(s) Of Engagement Activity 2013
 
Description NESCI Research Day 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact keynote lecture entitled "disease modelling with iPSC"

no notable impacts
Year(s) Of Engagement Activity 2014
 
Description Newcastle University Molecular Biosciences Research day 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Discussions resulted in more possible collaborations

No notable impacts so far
Year(s) Of Engagement Activity 2014
 
Description Presentation to Aptus Clinical,Manchester, 9th February 2017" Reprogramming drug discovery and development - the science of induced pluripotent stem cells" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact increasing industrial interest in use of stem cells for drug discovery
Year(s) Of Engagement Activity 2017
 
Description Presentation to Cardiff University, 8th July 2016, Induced Pluripotent Stem Cell modelling of cardiac disease" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact presentation and discussion of the application of pluripotent stem cells to modelling congenital heart abnormailities
Year(s) Of Engagement Activity 2016
 
Description Presentation to Charles River, Edinburgh, 7th February 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact discussion about using pluripotent stem cells to replace animal models of drug toxicity
Year(s) Of Engagement Activity 2017
 
Description Presentation to Covance Ltd, 24th February 2016, "development of iPSC based models of human skin" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact discussion about how we can use iPSC to build full thickness models of human skin
Year(s) Of Engagement Activity 2016
 
Description Presentation to Domainex, Ltd, 2nd March 2016, "iPSC for disease modelling" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact promoting iPSC for disease modelling
Year(s) Of Engagement Activity 2016
 
Description Presentation to Eurodiagnostica, Malmo, Sweden, 14th December 2016, Reprogramming drug discovery and development 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact investigating the utility of pluripotent stem cells as diagnostic tools in medicine
Year(s) Of Engagement Activity 2016
 
Description Presentation to ISCF Medicines Manufacturing Workshop, 10th August 2017, Newcastle University, UK, "Accelerating drug discovery" 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact presented ideas contributing to the creation of a drug discovery hub within Newcastle University
Year(s) Of Engagement Activity 2017
 
Description Presentation to Imanova, Ltd, 18th February 2016, iPSC for disease modelling" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact demonstrating the use of stem cells in drug discovery
Year(s) Of Engagement Activity 2016
 
Description Presentation to Masaryk University, 12th May 2017, Brno, Czech Republic, "Induced Pluripotent stem cell modelling" 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact presented data on stem cell based disease modelling with the purpose of inclusion into a Horizon 2020 programme grant
Year(s) Of Engagement Activity 2017
 
Description Presentation to ProQR therapeutics, "Production of Retinal and Otic vesicle Organoids from iPSC, 3rd May, Leiden, The Netherlands 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact presented the advantages of using retinal and otic organoids for the development of therapies for retinal and cochlear dystrophies
Year(s) Of Engagement Activity 2019
 
Description Presentation to Solvo Biotechnology, 27th October 2017, Newcastle, UK "prospects for iPSC based transporter assays" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact highlighted the possibility of using induced pluripotent stem cell derived cells to measure organic ion transporter activity
Year(s) Of Engagement Activity 2017
 
Description Presentation to StemBANCC general assembly, Chilly-Mazarin, Paris, France, "Pluripotent stem cell based models" 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact discussion / update of investigations into the utility of pluripotent stem cells as tools to model human disease
Year(s) Of Engagement Activity 2016
 
Description Presentation to StemBANCC general assembly, September 2017, Barcelona Spain, "Concluding data from the StemBANCC project" 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact presented the final data for the stembancc project which was aimed at generating a bank of 1500 iPSC lines from neurodegenerative disease patients
Year(s) Of Engagement Activity 2017
 
Description Presentation to UK Biobank, Stockport, UK, 26th February "Generation and characterisation of iPSC from small volumes of cryopreserved peripheral blood" 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact discussed how we might be able to expand the impact of the resources held in the UK biobank by deriving pluripotent stem cells
Year(s) Of Engagement Activity 2016
 
Description Presentation to Unilever PLC, Safety and Environmental Assurance Centre, 30th June 2017, Colworth, UK, "modelling disease and toxicology with induced pluripotent stem cells" 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Presentation to highlight the utility of stem cell derived in vitro models for toxicity testing
Year(s) Of Engagement Activity 2017
 
Description Presentation to in3 Workshop and 4th Network meeting, University of Natural Resources, Department of Biotechnology and Life Sciences, "Newcells Biotech - Concept, Launch and Beyond" 20th May, Vienna, Austria 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Workshop intended to educate academics, postgraduates and undergraduates about the process of creating a spin out company
Year(s) Of Engagement Activity 2019
 
Description StemBANCC consortium meeting, Boehringer Ingelheim, Biberach, Germany 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact decisions reached regarding application of induced pluripotent stem cells in the European pharmaceutical industry

no actual impacts realised to date
Year(s) Of Engagement Activity 2014
 
Description StemBANCC consortium meeting, Janssen Pharmaceuticals, Beerse, Belgium 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact presented data obtained during stembannc project but this led to important discussions about the requirements of the European Pharmaceutical industry for cells to be used in drug screening

none yet
Year(s) Of Engagement Activity 2013
 
Description Takeda Science Foundation Symposium on Pharmaceutical Sciences 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact the outcome of this talk was increasing the integration of my research group into the international stem cell community with some possible leads to future international collaborations

no impacts yet
Year(s) Of Engagement Activity 2014
 
Description Using human pluripotent stem cells to understand and treat inherited diseases 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited Lecture at the Annual meeting of the British Society of Human Genetics Conferences

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
 
Description Webinar delivered jointly with Evotec, 5th December 2017, Cambridge Healthcare Institute, New York, USA, "Pluripotent stem cells for drug discovery and toxicity assessment" 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Webinar used to promote the use of induced pluripotent stem cell disease models as drug discovery tools
Year(s) Of Engagement Activity 2017
 
Description modelling disease and toxicology with induced pluripotent stem cells 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Presentation to Unilever PLC, Safety and Environmental Assurance Centre, 30th June 2017, Colworth, UK
Year(s) Of Engagement Activity 2017
 
Description nternational Society for Stem Cell Research (ISSCR), Boston MA, USA. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact invited presentation on induced pluripotent stem cell banking to ISSCR
Year(s) Of Engagement Activity 2012