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Quantitative mapping of the proteomes of therapeutic stem cells.

Reference	BB/J021407/1
Principal Investigator / Supervisor	Professor Peter Walter Andrews
Co-Investigators / Co-Supervisors
Institution	University of Sheffield
Department	Biomedical Science
Funding type	Research
Value (£)	273,824
Status	Completed
Type	Research Grant
Start date	01/03/2012
End date	30/06/2014
Duration	28 months

Abstract

The key objective of the project is to develop tools for rapidly identifying when ES cell cultures acquire, during scale up for biomedical applications, genetic or epigenetic changes that might compromise their safe and effective use. We will provide to the consortium cultured adapted human ES cell lines exhibiting altered growth and differentiation patterns, and their early passage non-adapted counterparts. These lines will include already well characterised sets including normal/abnormal pairs of the human ES cell lines Shef4, Shef5, H1, H9 and H14. These typically have karyotypic changes but we also have stocks of late passage ES cells that have acquired altered growth patterns but without overt karyotypic change, perhaps due to sub-karyotypic genetic change or to epigenetic change. These and other lines provided by Neusentis will be assessed for various genotypic and phenotypic parameters of culture adaptation: genotypic change will be assessed by G-banding karyology and the presence of small populations of cells with gains of specific chromosomal regions will be assessed by interphase FISH. Analysis of copy number variation will also be performed when appropriate. Phenotypic analyses will include, as appropriate, the use of high content clonogenic assays to test for changes in the capacity of cells for survival, proliferation and differentiation, and time lapse microscopy will be used to assess cell motility parameters. Cell extracts will be prepared from normal and culture adapted sublines from the panel and provided to Cellzome for proteomic analysis. For specific experiments, cells will be sorted for expression of key stem cell markers such as SSEA3, SSEA4 and TRA-1-60, before sample preparation, to address the potential problem of cell heterogeneity caused by spontaneous differentiation. Proteomic differences that are then detected in culture adapted pairs of cells will be correlated with other geneotypic or phenotypic changes that are identified.

Summary

1. Provision of curated cultured adapted human ES cell lines and their early passage non-adapted counterparts. From existing panels of human ES cell lines we will curate and characterise sets of normal and culture adapted sublines that exhibit altered growth and differentiation characteristics. These will include sets that we have already well characterised, e.g. from the H7 human ES cell line, as well as others that we have banked, including normal/abnormal pairs of the human ES cell lines Shef4, Shef5, H1, H9 and H14. Many of these involve karyotypic changes. However, we also have stocks of late passage ES cells that have acquired altered growth patterns but with out overt karyotypic change, perhaps due to sub-karyotypic genetic change or to epigenetic change. These together with additional lines provided by Neusentis, grown under defined culture conditions, will also be assessed for various parameters of culture adaptation (see below). Cell extracts will be prepared from normal and culture adapted sublines from the curated panel and provided to Cellzome. Key parameters will be assessed on cultures corresponding to those provided to Cellzome, to ensure comparability and to facilitate correlation analyses. 2. Parameters of Culture Adaptation The properties of the cells will be analysed for genotype and phenotype, and by high content clonogenic assays for survival, proliferation and differentiation, and time lapse microscropy to assess cell motility parameters. By comparison of these with data generated by the Cellzome platform we will identify criteria that will provide the most robust indictors of culture adaptation. Genotyping: Cell lines will be assessed by standard G-Banding karyology. The presence of small populations of cells with gains of specific chromosomal regions will be assessed by interphase FISH. We will also assay the genotype of the cells using a Illumina1M Quad BeadChip, the same platform as used in the ISCI-2 genetic stability study, which willallow cross comparison of the cell lines in our study with over 120 other human ES lines whose karyotype and CNV status is known and with reference CNV data to enable the identification of culture induced CNV's. Phenotype: All human ES cells will be assessed for the expression of the panel surface antigens that are characteristic of human ES cells. Since these markers identify the undifferentiated cells, they can identify the extent of spontaneous differentiation in a culture of ES cells, a parameter that may change in concert with culture adaptation. Gene expression patterns will also be assessed using the TaqMan Low Density Array Q.PCR system designed for the International Stem Cell Initiative. This array includes key markers of undifferentiated human ES cells and their differentiated derivatives; in conjunction with surface antigen data these data will provide further indicators of culture adaptation. For example, we have found evidence that some cases of culture adaptation is characterised by loss of an ability to generate specific cell lineages, notably endoderm. High content clonogenic assays for survival, proliferation and differentiation: Clonogenic assays of human ES cells will be analysed using the GE Healthcare InCell Analyzer. Assessment of numbers of colonies, size of colonies and phenotype of cells within colonies wil provide detailed information about the growth properties of ES cells including their growth rate, ability to survive culture stress and their propensity for differentiation, including their pathways of differentiation. Cell motility: We have recently observed that the survival and motility of normal and culture adapted human ES cells under marked changes when observed by time lapse microscopy of single cells and their progeny. These parameters will be assessed for all sublines provided for Cellzome analysis.

Impact Summary

This project research aims to provide a robust platform for readily assessing the state of human pluripotent setm cell cultures with respect to culture adaptation. Although the development is focused on ES cells it is expected to be directly relevant to iPS cells, which generally closely mimic ES cells in their pluripotent properties and are subject to the same changes in behaviour, termed culture adaptation. Culture adaptation encompasses a range of changes in the growth and differentiation properties of human ES and iPS cells that may arise from the selection of variant cells with altered genotype or epigenotype on prolonged maintenance in culture. It is likely to be an inevitable, though stochastic, feature of maintaining and expanding cultures of these cells and may have considerable consequences for their eventual applications in regenerative medicine, or in drug screening and toxicology. Nevertheless detection and assessment of genetic and epigenetic changes associated with culture adaptation is not simple and it remains unclear how the different features are correlated. The current project aims to provide a robust and simple platform that will enable users of human ES and iPS cells to assess the quality of their cell lines as they are grown and expanded for their specific applications. It is anticipated that key users of the technology will be companies developing applications in regenerative medicine, in which ES, or iPS, cells are expanded and caused to differentiate into specific cell types for transplantation to patients. Examples include current trials by Geron Corp. in the USA for spinal cord injury, and ACT Corp. for Stargat's disease. Other forthcoming trials include potential applications in diabetes in the USA (Viacell Inc) or in the UK where trials are planned for treating Age Related Macular Degeneration. Other types of application in regenerative medicine are on the horizon. In all cases, culture adaptation of ES cells may reduce the efficiencyby which specific differentiated derivatives may be produced, while also causing potential safety problems since culture adapted cells may lose efficacy or acquire tumorigenic properties. Since culture adaptation make inevitably occur during expansion and production of cells for clinical application, any company involved in this area will need tools to closely monitor the state of their cell cultures and ensure early detection of culture adaptation. The regulatory authorities may also be expected to require information on the extent and nature of culture adaptation in cells to be used for regenerative medicine applications. It is expected that the current research will generate a platform to provide data that will be used by the regulatory agencies in assessing the safety of regenerative medicine products. Many companies in the pharmaceutical sector are likely to employ assays based on human ES and iPS cells in both toxicology and drug discovery. For example, in the UK, Stem Cells 4 Safer Medicine is a public private partnership company developing tools derived from human ES cells for hepatocyte and cardiomyocyte toxicology. In this areana as in regenerative medicine, it will be essential to continually monitor ES cells for signs of culture adaptation that may interfere with the production or efficacy of the derived hepatocytes or cardiomyocytes in toxicology assays.

Committee	Research Committee A (Animal disease, health and welfare)
Research Topics	Industrial Biotechnology, Stem Cells
Research Priority	X – Research Priority information not available
Research Initiative	Innovate UK (TSB) [2011-2015]
Funding Scheme	X – not Funded via a specific Funding Scheme

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