Quantitative mapping of the proteomes of therapeutic stem cells.

1.提供经过精选的培养适应的人类ES细胞系及其早期传代的非适应对应物。从现有的人类ES细胞系的面板中，我们将筛选和鉴定一组表现出改变的生长和分化特性的正常和培养适应的亚系。这些将包括我们已经很好地描述的集合，例如来自H7人类ES细胞系的集合，以及我们已经储存的其他集合，包括人类ES细胞系Shef4、Shef5、H1、H9和H14的正常/异常配对。其中许多涉及核型变化。然而，我们也有一些后期传代的ES细胞已经获得了改变的生长模式，但没有明显的核型改变，可能是由于亚核型遗传改变或表观遗传改变。这些以及Neusentis提供的在特定培养条件下生长的其他品系也将被评估各种培养适应参数(见下文)。细胞提取物将从正常的和培养适应的亚系中制备，并提供给Cellzome。将在与提供给Cellzome的培养相对应的培养上评估关键参数，以确保可比性并促进相关性分析。培养适应参数将对细胞的特性进行基因型和表型分析，并通过高含量克隆形成试验进行存活、增殖和分化，以及时间推移显微扫描来评估细胞运动参数。通过将这些数据与Cellzome平台产生的数据进行比较，我们将确定提供最可靠的培养适应指标的标准。基因分型：细胞系将通过标准的G显带核学进行评估。间期FISH将评估获得特定染色体区域的小群体细胞的存在。我们还将使用Illlighta1M四芯片检测细胞的基因型，该平台与ISCI-2遗传稳定性研究中使用的相同平台，将允许将我们研究中的细胞系与其他120多个已知核型和CNV状态的人类ES细胞系进行交叉比较，并参考CNV数据，以便能够识别培养诱导的CNV。表型：将评估所有人类ES细胞的面板表面抗原的表达，这些抗原是人类ES细胞的特征。由于这些标记识别未分化的细胞，它们可以识别ES细胞培养中自发分化的程度，这一参数可能会随着培养适应的变化而改变。基因表达模式也将使用为国际干细胞倡议设计的TaqMan低密度阵列Q.PCR系统进行评估。这个阵列包括未分化的人类ES细胞及其分化的衍生物的关键标记；结合表面抗原数据，这些数据将提供进一步的培养适应指标。例如，我们发现有证据表明，某些培养适应案例的特征是丧失产生特定细胞系的能力，尤其是内胚层。高含量的克隆细胞存活、增殖和分化分析：人类ES细胞的克隆形成分析将使用GE Healthcare Incell Analyzer进行。对集落数量、集落大小和集落内细胞表型的评估将提供有关ES细胞生长特性的详细信息，包括它们的生长速度、在培养压力下的生存能力和分化倾向，包括它们的分化途径。细胞运动性：我们最近观察到，通过单细胞及其后代的时间推移显微镜观察，正常和培养适应的人类ES细胞的存活率和运动性明显变化。这些参数将对为细胞分析提供的所有子线进行评估。

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.