Culture Adaptation in Human Embryonic Stem Cell Lines

Lead Research Organisation: University of Sheffield
Department Name: Biomedical Science

Abstract

Human embryonic stem (ES) cells offer considerable opportunities for regenerative medicine, drug discovery and toxicology. To realise this potential, a detailed understanding of their basic biology is required. It has become evident over the past three to four years that human ES cells may undergo genetic and other changes that may enhance their capacity for growth in culture, when they are maintained for extended periods. We have called this process, ‘culture adaptation’. The underlying hypothesis of this proposal is that the mechanisms affected during culture adaptation of ES cells are those that control the balance between self renewal, differentiation and death of these pluripotent stem cells. Understanding the basis for adaptation may not only provide a rational basis for designing improved methods to culture these cells while minimising the appearance of variants, but also may provide insights into the processes that control the self renewal of pluripotent stem cells and suggest approaches for controlling their growth and differentiation. Further, an understanding of culture adaptation of ES cells may provide insights into the mechanisms that drive progression of teratocarcinomas, a rare tumour of young men, but also a paradigm for other stem cell based malignancies.

Technical Summary

Human embryonic stem (ES) cells may undergo karyotypic changes upon prolonged culture. These genetic changes often accompany phenotypic changes that render the cells more robust and easier to maintain, a phenomenon termed ?culture adaptation?. This phenomenon is complex and may include alterations in cell fate decisions, as well as changes in proliferation rate, cloning efficiency, and the phenotype of cells with respect to specific markers, differentiation capacity and malignant potential. The karyotypic changes associated with adaptation are non-random, with acquisition of extra copies of chromosomes 17, 12 and X being especially common. These changes parallel similar genomic amplifications in embryonal carcinoma (EC) cells, a cancer counterpart of ES cells, which highlights the potential link of culture adaptation in ES cells to the development of a malignant phenotype. However, little is known about the factors that drive culture adaptation, nor the cellular and genetic mechanisms involved. Nevertheless, the appearance of such adaptive changes in human ES cells is likely to have a substantial impact upon their eventual application, whether in regenerative medicine or drug discovery and toxicology. In this project we will first define the features that characterise the culture adapted phenotype of human ES cells and the extent to which these features occur independently or in a co-ordinated fashion. We will then seek to determine the nature of the selective pressures that drive the appearance of variant ES cells and to establish how interaction of the different features of the culture adapted phenotype with culture and passage conditions provide cells with a selective advantage. Finally we will aim to identify in more detail the specific genetic changes and individual genes that contribute to the culture adapted phenotype of human ES cells.

Publications

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Barbaric I (2010) High-content screening of small compounds on human embryonic stem cells. in Biochemical Society transactions

 
Description Continuation of collaboration with The Radium Hospital, Oslo 
Organisation Institute of Cancer Research UK
Country United Kingdom 
Sector Academic/University 
PI Contribution Exchange of culture techniques in growing human ES cells, growth and sorting of human ES and EC cells. Transfer of cells from this group to Oslo to allow them to analyse gene expression.
Collaborator Contribution Transcriptional analysis of human ES cells and humn EC cells.
Impact Manuscript in preparation.
Start Year 2006
 
Description Joint PhD Studentship with A Star, Singapore 
Organisation Agency for Science, Technology and Research (A*STAR)
Country Singapore 
Sector Public 
PI Contribution Analysis of karyotype of variant hESC and analysis of data.
Collaborator Contribution Preparation and analysis of variant hESC lines, expressing genes from a minimal amplicon on chromosome 20q.
Impact Paper in preparation
Start Year 2010
 
Description Nuclear organisation and structure of hES cells. 
Organisation University of Bradford
Department School of Life Sciences Bradford
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of human ES cells for analysis.
Collaborator Contribution Sharing of technical expertise. Talk given to this group.
Impact Ongoing.
Start Year 2010
 
Description ESTOOLS Public Meeting, Rome 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Public meeting open to lay public, journalists and policy makers as well as scientists

Contributed to discussion in Italy about research with human ES cells
Year(s) Of Engagement Activity 2008
 
Description ESTOOLS public meeting, Lisbon 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact 300 delegates were brought together at the final ESTOOLS annual meeting. In addition, there was a theatre event open to members of the public which generated interest, questions and debate in the use of human embryonic stem cell research within Europe.

Unknown.
Year(s) Of Engagement Activity 2010
 
Description Researchers Night 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Type Of Presentation Poster Presentation
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact A night to attract and engage the public in the work of the Centre for Stem Cell Biology. Attended by over 200 members of the public. A range of activities were offered; including screenings of short films, informative posters, interactive games and practical demonstrations.

Excellent feedback received back from the vast majority of attendees. Will write a blog describing the event for Eurostemcell who provided some of the outreach materials.
Year(s) Of Engagement Activity 2012