Abstract
6555 Background: The tracking of stem cell aging, differentiation and deterioration by gene expression profiling and proteome analysis allows the comparison of different stages. The overall aim of a proteomic study is characterization of the complex network of cell regulation. We focused our investigations on different subsets of highly enriched CD34+ stem cells from different human origins: fetal liver, cord blood, bone marrow (BM), and mobilized stem cells from peripheral blood (PBSC), as well as CD34+ leukemia cells, thus e.g. to identify pathways and new targets for leukemia therapy. Methods: Mononuclear cells were isolated by a standard Ficoll separation method from the different blood sources. An Auto-MACS (Miltenyi) and FACS Vantage SE cell sorter (Becton Dickinson) was used to highly enrich (>99%) CD34+ cells fractions. Sample preparation: Total RNA was isolated from sorted 1 × 10e6 cells by standard methods using RNA isolation kit (Qiagen). For gene expression analysis topic-defined PIQOR stem cell microarrays (936 genes) were performed. Proteomics started with the determination of protein concentrations, 2D-gel-electrophoresis were described in Proteome Works System (BioRad). Sypro ruby and/or coomasie stained gels were used for protein identification by Q-TOF analyses. The subcellular localization of the identified proteins were performed by fluorescence and confocal microscopy of all cell fractions. Results: 1. The microarray gene expression correlation shows many similarities between human healthy stem cells of different sources and ages, otherwise many differences: 125 upregulated genes (kinases: PAK1, ATM1, CDKN2A) and 32 downregulated genes (EBCTF, RAMP1) in malignant cells compared to healthy stem cells. 2. Proteomics analyses of the different cell fractions show a large overlap of the most dominant protein spots, >200 spots were identified by Q-TOF. For example stathmin (oncoprotein Op18) is expressed at very high levels in leukemia cells and in PBSCs but not in BM cells, additionally demonstrated by fluorescence microscopy. Conclusions: Combining genomics and proteomics assays, pathways e.g. Op18 for proliferation and migration of healthy (mobilized) CD34+ cells from bone marrow and malignant leukemia cells could be identified. No significant financial relationships to disclose.
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