gms | German Medical Science

Aim: 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: cord blood (CB), 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-Hypaque gradient 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 x 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 (e.g. kinases: PAK1, ATM1, CDKN2A) and 32 downregulated genes in malignant cells compared to healthy stem cells. 2. The protein pattern of the CD34+ highly enriched stem cell fractions is complex and shows >1000 protein spots (dependent on protein concentration and staining) using PDQuest software. 3. The most dominant protein spots (>100) were cutted and identified by Q-TOF. There is a strong overlap between the detected major proteins in all analyzed cell fractions. 4. E.g. 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: Using Genomics and Proteomics assays, pathways (substrate, kinases) e.g. for proliferation and migration of healthy (mobilized) CD34+ cells from bone marrow and malignant leukemia cells could be identified.