Genomic and Proteomic Signatures of Human Mesenchymal Stem Cells.
Authors: Wagner Wolfgang , Feldmann Robert E. , Wein Frederik , Seckinger Anja , Krause Ulf , Frankhauser Maria , Maurer Martin H. , Blake Jonathon , Eckstein Volker , Saffrich Rainer , Wuchter Patrick , Kuschinsky Wolfgang , Ho Anthony D. 
Published: April 2020

Abstract
Mesenchymal stem cells (MSC) raise high hopes in clinical applications. However, the lack of common standards and a precise definition of MSC preparations remains a major obstacle in research and application of MSC. Whereas surface antigen markers have failed to precisely define this population, a combination of proteomic data and microarray data provides a new dimension for the definition of MSC preparations.
To define the molecular signatures at a genomic and proteomic level we have compared the MSC preparations isolated from different sources (adipose tissue, bone marrow and umbilical cord blood) from different donors, as well as from the same donor cultured under different conditions. As a references we have used non-multipotent human fibroblasts (HS68 and NHDF). No phenotypic differences were observed between MSC and fibroblasts by flow cytometry using a panel of 22 surface antigen markers. Gene expression profiles were compared by cDNA microarray analysis (51,144 different cDNA clones of the RZPD3 Unigene Set). Twenty-five genes were overlapping more than two-fold up-regulated in all MSC preparations and these included fibronectin, ECM2, glypican-4, ID1, NF1B, HOXA5 and HOXB6 whereas several inhibitors of the Wnt-pathway (DKK1, DKK3, SFRP1) were higher expressed in fibroblasts. Differential gene expression was verified for 20 genes by RT-PCR. Hierarchical cluster analysis revealed a correlation of four individual donor samples for each MSC preparation while pair wise comparison of MSC from different tissues or culture-isolation procedures revealed between 206 to 1113 differentially expressed ESTs (p&lt;0.001). Using two-dimensional gel electrophoresis and mass spectometry we have generated a proteome reference map of MSC. 136 protein spots were unambiguously identified most of which play a role in cytoskeleton, protein folding and metabolism. This reference map was used to compare protein expression of MSC isolated under different culture conditions. Corresponding cDNA clones were then selected on our microarray. Combination of datasets revealed that genes that were differentially expressed on mRNA level (p&lt;0.05) were differentially expressed on protein level as well (Pearson correlation = 0.83). Interchanging culture conditions revealed that differential expression was directly regulated by medium in some genes whereas it remained constant in others. Expressions of fibronectin, vimentin, myosin, tropomyosin and ezrin were analyzed by fluorescence microscopy whereby no subpopulations could be discriminated within cell preparations while marked differences were observed in morphology and organization of MSC isolated under different culture conditions.
Our results provide evidence for reproducible isolation of similar and homogeneous MSC preparations under standardized isolation conditions, while MSC from different tissues or culture conditions display significant differences in their transcriptome, proteome and cellular organization. Our comparative approach provides foundation for a reliable quality control using genotypic and proteomic analysis for clinical applications.