Quantitative mapping of the proteomes of therapeutic stem cells.

点击翻译按钮获取中文摘要

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 sublinesthat exhibit altered growth and differentiation characteristics. These will include sets that we have already wellcharacterised, e.g. from the H7 human ES cell line, as well as others that we have banked, including normal/abnormal pairsof the human ES cell lines Shef4, Shef5, H1, H9 and H14. Many of these involve karyotypic changes. However, we alsohave 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 byNeusentis, grown under defined culture conditions, will also be assessed for various parameters of culture adaptation (seebelow). Cell extracts will be prepared from normal and culture adapted sublines from the curated panel and provided toCellzome. Key parameters will be assessed on cultures corresponding to those provided to Cellzome, to ensurecomparability and to facilitate correlation analyses.2. Parameters of Culture AdaptationThe properties of the cells will be analysed for genotype and phenotype, and by high content clonogenic assays forsurvival, proliferation and differentiation, and time lapse microscropy to assess cell motility parameters. By comparison ofthese with data generated by the Cellzome platform we will identify criteria that will provide the most robust indictors ofculture adaptation.Genotyping: Cell lines will be assessed by standard G-Banding karyology. The presence of small populations of cells withgains of specific chromosomal regions will be assessed by interphase FISH. We will also assay the genotype of the cellsusing a Illumina1M Quad BeadChip, the same platform as used in the ISCI-2 genetic stability study, which will allow crosscomparison of the cell lines in our study with over 120 other human ES lines whose karyotype and CNV status is knownand 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 ofhuman ES cells. Since these markers identify the undifferentiated cells, they can identify the extent of spontaneousdifferentiation in a culture of ES cells, a parameter that may change in concert with culture adaptation. Gene expressionpatterns will also be assessed using the TaqMan Low Density Array Q.PCR system designed for the International StemCell Initiative. This array includes key markers of undifferentiated human ES cells and their differentiated derivatives; inconjunction with surface antigen data these data will provide further indicators of culture adaptation. For example, we havefound evidence that some cases of culture adaptation is characterised by loss of an ability to generate specific celllineages, notably endoderm.High content clonogenic assays for survival, proliferation and differentiation: Clonogenic assays of human ES cells will beanalysed using the GE Healthcare InCell Analyzer. Assessment of numbers of colonies, size of colonies and phenotype ofcells 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 undermarked changes when observed by time lapse microscopy of single cells and their progeny. These parameters will beassessed for all sublines provided for Cellzome analysis.