Molecular mechanisms controlling differentiation of pluripotent cells into endoderm.

Lead Research Organisation: University of Cambridge
Department Name: Surgery

Abstract

During the last decade various stem cells have been derived from adult or embryonic human tissues. These cells offer new prospects to treat degenerative diseases, since they can specialise into many mature cell types that could be useful for cell based therapies. However, robust protocols allowing the production of fully functional specialised cells still need to be established. The generation of such cell types may ultimately only be achievable by recapitulating a normal path of development in vitro. But the sequential events leading to the formation of the primary germ layers, the building block of the body‘s organs, remain obscure. Thus, the understanding of the mechanisms controlling the specification of the primary germ layers has a major importance for regenerative medicine. Here, I propose to use human Embryonic Stem cells (hESCs) to study the mechanisms controlling differentiation of the endoderm germ layer, from which originate the pancreas, liver, lungs, and gut. While the objective of this project is to control differentiation of hESCs into endoderm lineages, the insight that will be gained could also be used to convert adult cells to endodermal cells by recapitulating the developmental pathway that forms this tissue during normal development.

Technical Summary

Generation of all the adult tissues occurs by progressive cell fate decisions, starting with the specification of the three primary germ layers (ectoderm, mesoderm and endoderm). Molecular mechanisms controlling specification of the germ layers have been extensively studied in amphibia and fish. However, the understanding of these mechanisms in mammals (especially humans) is more limited, and this represents a major drawback for regenerative medicine. Indeed the generation of fully functional cell types from stem cells may only be achievable by recapitulating a normal species specific succession of cell fate decisions.
The studies of these mechanisms at the molecular level in vivo are restricted in mammals and particularly in humans, by the difficulty of obtaining sufficient biological material. The recent availability of human Embryonic Stem cells (hESCs) offers new possibilities to resolve this major limitation. Their embryonic origin confers upon them the ability to proliferate indefinitely in vitro while maintaining the capacity to differentiate into extra-embryonic tissues and into derivatives of the three primary germ layers. Here, I propose to define the network of transcription factors controlling differentiation of pluripotent cells into endoderm using first hESCs as an in vitro model of human development and then using mouse embryos to validate these results in vivo.
This project will be based on functional studies and on high throughput approaches including chromatin immunoprecipitation (ChIP) assays and gene expression profiling. Importantly, differentiation of hESCs into definitive endoderm cells will be achieved using fully chemically defined culture conditions which are devoid of animal products, thereby eliminating factors that could obscure analysis of developmental mechanisms or render the resulting tissues incompatible with future clinical applications. This method of differentiation has been developed during my current fellowship and the endoderm progenitors generated have been extensively characterised by both molecular analytical and cellular functional assays.
In summary, the objectives of this proposal are (i) to identify the key transcription factors controlling endoderm differentiation and to define precisely their function, (ii) to reveal the organisation of the core transcriptional network controlling the expression of these transcription factors during the transition between the pluripotent state and the endoderm fate and (iii) to define the downstream network of genes controlled by these transcription factors. The overall outcome of this project will gain the knowledge needed to control differentiation of hESCs into specific lineages and also potentially to enable trans-differentiation of other cell types, including adult stem cells and adult somatic cells.

Publications

10 25 50
 
Description International consortium large scale hIPSCs project
Geographic Reach Multiple continents/international 
Policy Influence Type Participation in a national consultation
 
Description European Research Council - Reprogramming cell identity to develop new therapies against Inherited Metabolic Disorders of the liver
Amount £1,373,000 (GBP)
Organisation European Research Council (ERC) 
Sector Public
Country European Union (EU)
Start 12/2011 
End 11/2016
 
Description Evelyn Trust - In vitro modelling of lung diseases using human Induced Pluripotent Stem Cells
Amount £145,366 (GBP)
Organisation The Evelyn Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2011 
End 08/2013
 
Description Innovative strategies to generate human hepatocytes for treatment of metabolic liver diseases: tolls for personalized cell therapy.
Amount £450,000 (GBP)
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 12/2011 
End 11/2014
 
Description Liv-ES Development of culture conditions for the differentiation of hES cells into hepatocytes (3m euros across 7 participants)
Amount £180,000 (GBP)
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 12/2008 
End 11/2012
 
Description MRC/Wellcome Trust Strategic award
Amount £12,500,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2012 
End 09/2016
 
Description Manufacturing Solution for high value Induced pluripotent stem cells product
Amount £656,986 (GBP)
Organisation Innovate UK 
Sector Public
Country United Kingdom
Start 09/2010 
End 08/2013
 
Description The production of a 3D human tissue disease platform to enable regenerative medicine therapy development
Amount £570,000 (GBP)
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 01/2012 
End 12/2015
 
Title Foregut Stem Cells 
Description We have identified a new type of endodermal stem cells. 
Type Of Material Cell line 
Year Produced 2014 
Provided To Others? Yes  
Impact These cells could enable the production of homogenous population of pancreatic, lung, and hepatic cells from a broad number of hPSCs. 
 
Title Human endoderm cells and derivatives 
Description We sdeveloped a new culture system to differentiate human pluripotent stem cells into endoderm and then into pancreatic and hepatic cells. 
Type Of Material Technology assay or reagent 
Year Produced 2007 
Provided To Others? Yes  
Impact These cultures systems are fully defined and fully compatible with clinical applications 
 
Title Protocol to generate pancretaic cells from hPSCs 
Description We have developed a new protocol to generate pancreatic cells from hPSCs in defined conditions. 
Type Of Material Technology assay or reagent 
Year Produced 2011 
Provided To Others? Yes  
Impact Important new information regarding pancreatic development and future clinical applications 
 
Title hIPSCs lines from patient with metabolic diseases 
Description We have derived a lrge number of hIPSCs lines from patients suffereing of diverse diseases 
Type Of Material Cell line 
Year Produced 2009 
Provided To Others? Yes  
Impact We hope to develop new in vitro of model disease for basic studies and drug screening. 
 
Title improve efficiency of Chromatin IP 
Description We developed an new method to perform ChIP -seq on factor which does not bind DNA directly such as Smad2/3. 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact This approach have allowed to map Smad2/3 binding sites at the genome wide level and is now currently used by a broad number of groups to perform similar studies on a diversity of transcription factors. 
 
Description Cardiovascular diseases 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution Our objective is to generate hIPSCs from patient with cardio-vascular disorders
Collaborator Contribution Expertise in disease model and sharing reagents
Impact My group was involved in a grant application related to this project and we are planning to train several collaborators.
Start Year 2009
 
Description Differentiation of Pancreatic cells 
Organisation University of Aberdeen
Department College of Life Sciences and Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution We are currently developing protocol to generate pancreatic cells from human pluripotent stem cells for clinical applications.
Collaborator Contribution We published two publications and we have applied for several grants
Impact We published two artciles and applied successfully to several grants. PubMedID 19056911 17959396
Start Year 2006
 
Description Encapsulation of pancreatic cells 
Organisation Norwegian University of Science and Technology (NTNU)
Department Department of Cancer Research and Molecular Medicine (IKM)
Country Norway 
Sector Academic/University 
PI Contribution The main goal is to encapsulate pancretaic cells from hESCs or hIPSCs and to define the functionality of the resulting cells in animal model
Collaborator Contribution Eu grant application + complementary expertise
Impact Eu grant application plus training and visit. 1 publication
Start Year 2011
 
Description Endoderm Differentiation 3 
Organisation Agency for Science, Technology and Research (A*STAR)
Department Genome Institute of Singapore
Country Singapore 
Sector Academic/University 
PI Contribution Our obejctive is to study the mechanisms controlling endoderm differentiation.
Collaborator Contribution We co-supervise a PhD a ASTAR PhD student
Impact We have published one publication and two others are in preparation.
Start Year 2008
 
Description Epigenetic status of pancreatic progenitors generated from hESCs 
Organisation August Pi i Sunyer Biomedical Research Institute
Department Endocrinology Unit
Country Spain 
Sector Multiple 
PI Contribution We have provided pancreatic progenitors generated from hIPSCs/hESCs
Collaborator Contribution Complementary expertise and network collaboratorsComplemtenary expertise and future grant applications
Impact 1 Publication and EU grant application have been submitted.
Start Year 2010
 
Description Epigenetic status of pancreatic progenitors generated from hESCs 
Organisation University of Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided pancreatic progenitors generated from hIPSCs/hESCs
Collaborator Contribution Complementary expertise and network collaboratorsComplemtenary expertise and future grant applications
Impact 1 Publication and EU grant application have been submitted.
Start Year 2010
 
Description Gene knock out in hPSCs 
Organisation The Wellcome Trust Sanger Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Perform gene targeting in hPSCs using TALENs provided by collaborator
Collaborator Contribution Provide and design TALENS
Impact None the collaboration was stopped due to technical difficulties
Start Year 2012
 
Description Generation of hepatocyte 
Organisation Hopital Bicetre
Country France 
Sector Hospitals 
PI Contribution Weare developing protocols to generate liver cells from human plurioptent stem cells
Collaborator Contribution We have published 3 manuscripts and applied for several grant including a sucessful EU grant.
Impact We have published 3 papers and obtained one EU-Grant Liv-ES PubMEDID: 19564924 1968839 Touboul et al., in press in Hepatology
Start Year 2007
 
Description Genetic of metabolic disoders 
Organisation University College London
Department Structural Molecular Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution Develop in vitro model of metabolic diseases especially affecting the liver
Collaborator Contribution Access to patients and disease expertise
Impact Grant applications
Start Year 2012
 
Description In vivo validation of IPSC derived hepatocytes 
Organisation École normale supérieure de Lyon (ENS Lyon)
Department INSERM U846 (Stem-cell and Brain Research Institute)
Country France 
Sector Academic/University 
PI Contribution Test capacity of hIPSC derived hepatocytes to colonise the liver of mouse model for liver failure
Collaborator Contribution Animal model + grant applications
Impact Publication + Grant application
Start Year 2010
 
Description In vivo validation of hIPSCs-hep 
Organisation Pasteur Institute, Paris
Country France 
Sector Academic/University 
PI Contribution In vivo validation of hIPSCs derived hepatocytes
Collaborator Contribution Animal model
Impact Unique expertise and animal models. Grant applications and publication
Start Year 2010
 
Description Mesoderm differentiation 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution Our main objective is to study human pluripotent stem cells.
Collaborator Contribution We have written several publications and applied for several grant.
Impact We have obatined several MRC grant and we ahve been co-author on a lrge number of publications
 
Description Pluripotency and Cancer 
Organisation Karolinska Institute
Country Sweden 
Sector Academic/University 
PI Contribution Our main objective is to use human pluripotent stem cells to study cancer.
Collaborator Contribution provide expertise on teratoma formation and perform experiments
Impact We have published one paper in collaboration and also applied for a grant. Pub Med ID:19688839
Start Year 2007
 
Description hESCs and training 
Organisation University of Liege
Country Belgium 
Sector Academic/University 
PI Contribution The mainobjective of this collaboration is to use hESCs as in vitro model of neuronal development.
Collaborator Contribution None
Impact We have provided training to grow hESCs and exchanging idead/information on a regular basis.
Start Year 2008
 
Description hIPSC differentiation into Cholangyocites 
Organisation King's College Hospital NHS Foundation Trust (NCH)
Department Paediatric Liver Centre
Country United Kingdom 
Sector Hospitals 
PI Contribution Bring expertise in hIPSC and protocol of differentiation toward cholangyocite
Collaborator Contribution Access to patients
Impact This collaboration has resulted in a successful application to a MRC clinical PhD fellowship and in 1 publication
Start Year 2013
 
Description hIPSCs and Epigenetic 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution The main objective of this colaboration is to define epigenetic variability among human induced pluripotent stem cells lines.
Collaborator Contribution Develop new approach to characterise hIPSCs
Impact Two publications are curentrly in preparation and we obatined a grant from the TSB to continue this work.
Start Year 2008
 
Description hPSCs and cell cycle 
Organisation University of Georgia
Country United States 
Sector Academic/University 
PI Contribution exchange of data and reagents
Collaborator Contribution exchange of data and reagents
Impact publications in preparation
Start Year 2013
 
Title Cell-Cycle Directed Differentiation of Pluripotent Cells 
Description Cell-Cycle Directed Differentiation of Pluripotent Cells 
IP Reference  
Protection Protection not required
Year Protection Granted 2013
Licensed No
Impact publication
 
Title Chemically defined culture conditions to drive differentiation of pluripotent cells into primary germ layers and into extra-embryonic tissues 
Description Chemically defined culture conditions to drive differentiation of pluripotent cells into primary germ layers and into extra-embryonic tissues 
IP Reference 00 09006 
Protection Patent application published
Year Protection Granted
Licensed Yes
Impact NA
 
Title In Vitro Pancreatic Differentiation of Pluripotent Mammalian Cells 
Description In Vitro Pancreatic Differentiation of Pluripotent Mammalian Cells 
IP Reference  
Protection Protection not required
Year Protection Granted 2013
Licensed Yes
Impact publications
 
Title In Vitro Production of Foregut Stem Cells 
Description derivation of foregut stem cells 
IP Reference  
Protection Protection not required
Year Protection Granted 2013
Licensed No
Impact publications
 
Title In vitro hepatic differentiation 
Description defined culture conditions to generate hepatocytes from hIPSCs for disease modelling 
IP Reference WO2012025725 
Protection Patent application published
Year Protection Granted 2012
Licensed Yes
Impact NA
 
Title Method to derive pluripotent cells from post-implantation stages 
Description Method to derive pluripotent cells from post-implantation stages 
IP Reference 07824531.3 
Protection Patent granted
Year Protection Granted
Licensed Yes
Impact NA
 
Title NUCLEAR REPROGRAMMING SUBSTRATE 
Description Method for generating hIPSC from Endothelial Progenitor Cells. 
IP Reference WO2012131387 
Protection Patent application published
Year Protection Granted 2012
Licensed Yes
Impact NA
 
Title Disease model for drug and toxicology screening 
Description Hepatocytes differentiated from hIPSC generated from patients with genetic disorders. 
Type Therapeutic Intervention - Drug
Current Stage Of Development Initial development
Year Development Stage Completed 2011
Development Status Under active development/distribution
Impact Creation spin out DefiniGEN 
 
Company Name DefiniGEN 
Description Definigen provides human liver cells for preclinical drug development and disease modelling applications using human Induced Pluripotent Stem Cell hIPSC technology. http://www.definigen.com/ 
Year Established 2012 
Impact NA
Website http://www.definigen.com/
 
Description BHF fund rising Cambridge 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Regional
Primary Audience Participants in your research and patient groups
Results and Impact 30 potential fund raisers

NA
Year(s) Of Engagement Activity 2013
URL http://www.bhf.org.uk/media/news-from-the-bhf/bhf-at-the-science-museum.aspx
 
Description Cambridge Science Festival 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact 80 person attended to the talk and the following discussion.

Very goof feedback from patients group.
Year(s) Of Engagement Activity 2011
 
Description Db UK / JDRF day in Cambridge 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Participants in your research and patient groups
Results and Impact Discussion panel concerning future therapy of diabetes

None
Year(s) Of Engagement Activity 2009
 
Description Diverse New letters of patient group 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact NA
Year(s) Of Engagement Activity 2011
 
Description House of lord enquiry on regenerative medicine 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Type Of Presentation Workshop Facilitator
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact NA / too soon

NA/ too soon
Year(s) Of Engagement Activity 2012
 
Description Night in the museum 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact around 100 people came to see the talk.

NA
Year(s) Of Engagement Activity 2013
URL http://www.bhf.org.uk/media/news-from-the-bhf/bhf-at-the-science-museum.aspx
 
Description Press release BHF EPC paper 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact No information available yet
Year(s) Of Engagement Activity 2012
 
Description Press release Nature publication 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact We had two press release (2010 and 2011) which resulted in more than 200 comments on radio, TV, web site, journals magazine, etc world wide

Very good return from the public and the stem cells field in general
Year(s) Of Engagement Activity 2010,2011