Directed differentiation of human embryonic stem cells to forebrain neural progenitors

Lead Research Organisation: Cardiff University
Department Name: School of Biosciences

Abstract

Embryonic stem cells, or ES cells, have the remarkable ability to differentiate into all cell types of the body. Controlled growth and differentiation of ES cells in the lab can be directed to generate large populations of specific types of cell. The advent of ES cells derived from human embryos has raised the exciting possibility that tissues required for human transplantation therapies could be grown in the lab, and circumvent the requirement for donor cells that are otherwise very difficult to source. This is particularly true neural transplantation that is currently being pioneered to treat disorders such as Parkinson?s and Huntington?s (HD) diseases, but is currently dependent on rare tissue, derived from elective aborted fetuses. One problem is that specific brain regions are characterized by the presence of different types of neuron, therefore to treat different disorders the right kind of neuron must first be generated. In this proposal we aim to control the differentiation of human ES cells to acquire the characteristic of the striatum, the region of the brain worst affected in HD, and the target for cell transplantation therapy. We will then test whether the derived cells will integrate and develop alongside host striatal tissue in animal models of HD.

Technical Summary

Human embryonic stem cells (hESC) have enormous potential to provide a reproducible and stable supply of cells for neural transplantation therapies that would otherwise not be possible due to a lack of primary donor fetal tissue. This is particularly so for treatment of neurological disorders such as Huntington?s disease (HD). Here we propose to extend our previous studies on the directed neural differentiation of mouse embryonic stem cells to hESCs, with the specific purpose of generating neural progenitor cells characteristic of the developing striatum, and testing their functional development using established transplantation paradigms in the rat. The work will involve the application of developmental principles of neural patterning and lineage restriction to pluripotent hESCs, drawing significantly from extensive knowledge of the mechanisms of brain development in the mouse and other model vertebrate organisms. This proposal addresses issues central to the purpose of the Stem cell strategic grants call; (i) the development of defined medium for directed stem cell differentiation (ii) the development and definition of progenitor cell type specific markers (iii) comparison of ES cell line differentiation characteristics (vi) development of protocols of significance to human therapy.

Publications

10 25 50
 
Description MRC Project Grant
Amount £700,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2008 
End 03/2012
 
Title Bicistronic Lentiviral iPS vectors 
Description Bicistronic lentiviral vectors were made for iPS cell generation. Oct4-IRES-Sox2 and Klf4-IRES-Nanog constructs were made in integrating and non-integrating virus 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Vectors have been used to derive human iPS cells. Establishing the technology within the group will allow us to further develop human models of neurodegenerative disease. 
 
Title Human ES cell neural differentiation methodology 
Description Development of chemically defined (to be GMP compliant) culture media and protocols for directed neural differentiation. Media avoid use of all animal products, and are used for efficient generationa nd expansion of neural progenitors. 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact The basic protocol is being further adapted to establish GMP compliant conditions to derive cells for neural transplantation therapies 
 
Title Neural disease modelling using human ES cells 
Description Differentiation protocols developed have been applied to ES cell derived from Huntington's Disease embryos do derive neurons for in vitro disease modelling of HD and development of cells for assay development and drug screens 
Type Of Material Model of mechanisms or symptoms - human 
Year Produced 2007 
Provided To Others? Yes  
Impact The research formed a basis for studies in the STEM-HD FP6 programme to develop human ES to model and study HD 
 
Description STEM-HD 
Organisation National Institute of Health and Medical Research (INSERM)
Department Institute for Stem cell Therapy and Exploration of Monogenic diseases (I-Stem)
Country France 
Sector Academic/University 
PI Contribution EU FP6 funded STREP. Stem cells for therapeutics and exploration of mechanisms in Huntingtons disease. Workpackage 1 leader: directed neural differentiation of human ES cells, to further develop differentiation protocols, particulalry for applications in high throughput screening. Gene expression profiling and analysis of wt and HD hESs and neural derivatives Physiological analysis of hES derived neurons
Collaborator Contribution Exchange of information and materials
Impact The project has established proof of concept for utilising disease carrying human embryonic stem cells derived from preimplantation diagnosed embryos for in vitro disease modelling. A platform for highthroughput drug screening using the human cells has been developed, and novel disease biomarkers have been identified. Publications 17095704 Other manuscripts in preparation
Start Year 2006
 
Description iPS technology 
Organisation University of Bristol
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided expertise in human ES cell culture, differentiation, and disease modelling
Collaborator Contribution Our collaborators have provided valuable expertise and reagents for lentiviral generation and neurodegenerative disease mechanisms
Impact We obtained supplementry funding to this award to develop iPS technology. This has now been established locally and is integral to future planned research programmes.
Start Year 2008
 
Description Laboratory open day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Together with the Brain Repair Group at Cardiff University we hold open days for HD and PD patient and support groups to inform them of ongoing research related to stem cells and brain repair

Direct communication with patient groups provides important support and hope.
Year(s) Of Engagement Activity 2006,2007,2008,2009
 
Description Public debate - Cardiff 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Public debate on stem cell medicine and ethics. Hosted by Techniquest Cardiff

Education of public on realistic issues and potentials of stem cell research
Year(s) Of Engagement Activity 2007