We characterized and compared the proteomic composition of ECM produced in vitro by bone marrow-derived MSC, adipose-derived MSC and neonatal fibroblasts isolated from different donors, employing a multidimensional proteomic approach coupled with label-based quantitative proteomics. Each cell-derived ECM displayed a specific and unique matrisome signature, yet they all shared a common set of proteins. We evaluated the biological response of cells cultured on the different matrices and compared them to cells on standard TCPS. The matrices lead to differential proliferation and gene expression profiles between the cell types and as compared to TCPS, indicating that the cell-derived ECM influence each cell type in a unique manner.