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U. Vossmerbaeumer, H.-J. Thierse, K. Bieback, S. Kuehl, I. Kreissig, J. B. Jonas; Differential Regulation of Proteins in Human Mesenchymal Stem Cells Following RPE Induction. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5783.
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Human Mesenchymal Stem Cells (MSC) have the potential to differentiate into a variety of cell types of the mesodermal lineage. In in-vitro conditions they can be induced to take a differentiation path beyond their natural lineage as shown for characteristics of retinal pigment epithel cells (RPE). The current study investigates to what extent up- or downregulation of specific proteins can be traced over the period of differentiation.
Human MSC were isolated from liposuction material and characterized for lineage specific undifferentiation properties. Transdifferentiation to RPE lineage was induced using porcine RPE conditioned medium (CM). Cells were lysed using standard proteomic lysis protocol (urea) and total protein was collected. All experiments were done with both pre- and postinduction material. Proteomic analysis was performed by classical and differential 2D gel electrophoresis (2DE, DIGE). Focusing was achieved with an IPGphor system and for 2-D separation the Ettan II Dalt system was used (GE Healthcare). Parallel assessment of the material was done with Flamingo staining and with standard DIGE analysis. The gels were analyzed in a Fuji FLA 5100 fluorescence scanner (Fuji) and data decoded by Delta2D software (Decodon). Subsequently, differential regulated proteins were subjected to mass spectrometric analysis.
Proteins were separated by isoelectrical focusing (pH 4-7L). 2DE comparison of protein expression patterns of native vs. induced MSC showed induction and inhibition for single proteins before and after the differentiation stimulus as visualized by distinct spots in the gels. Results from experiments with Flamingo stains are in line with DIGE analysis, thus providing complementary information. Proteomic pattern analysis revealed signals that were either detectable pre- or post induction. Identification of differentially regulated proteins was performed by mass spectrometry.
The experiments demonstrated that human MSC display a differential proteomic expression pattern with significant alteration after induction of RPE-directed differentiation. Current research includes identification of regulated proteins as found in DIGE-experiments using mass spectrometry. Besides established differentiation markers novel proteins apt to indicate transition in differential status might be identified.
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