Human bone marrow-derived multipotent mesenchymal stromal cells, often referred to as mesenchymal stem cells (MSCs), represent an attractive cell source for many regenerative medicine applications due to their potential for multi-lineage differentiation, immunomodulation, and paracrine factor secretion. A major complication for current MSC-based therapies is the lack of well-defined characterization methods that can robustly predict how they will perform in a particular in vitro or in vivo setting. Significant advances have been made with identifying molecular markers of MSC quality and potency using multivariate genomic and proteomic approaches, and more recently with advanced techniques incorporating high content imaging to assess highdimensional single cell morphological data. We sought to expand upon current methods of high dimensional morphological analysis by investigating whether short term cell and nuclear morphological profiles of MSCs from multiple donors (at multiple passages) correlated with long term mineralization upon osteogenic induction. Using the combined power of automated high content imaging followed by automated image analysis, we demonstrated that MSC morphology after 3 days was highly correlated with 35 day mineralization and comparable to other methods of MSC osteogenesis assessment (such as alkaline phosphatase activity). We then expanded on this initial morphological characterization and identified morphological features that were highly predictive of mineralization capacities (>90% accuracy) of MSCs from additional donors and different manufacturing techniques using linear discriminant analysis. Together, this work thoroughly demonstrates the predictive power of MSC morphology for mineralization capacity and motivates further studies into MSC morphology as a predictive marker for additional in vitro and in vivo responses. STEM CELLS 2016;34:935-947 SIGNIFICANCE STATEMENTThis article presents a new approach to assess the quality of mesenchymal stem cells (MSCs) for a given osteogenic assay, as well as a means to compare the effects of different culturing and isolation techniques on MSC behavior. This automated, high content imaging approach could be used to compare characteristics of MSC lots from different laboratories and potentially identify morphological signatures that effectively predict their performance in an osteogenesis bioassay. Furthermore, this article highlights the necessity for quantifying multiple osteogenic assay outcomes as simply gene expression and alkaline phosphatase activity alone were found to be highly variable and poorly correlated with more long term, mature MSC osteogenesis based on the extent of mineralization.

IntroductionHuman multipotent stromal cells (MSCs) isolated from bone marrow or other tissue sources have great potential to treat a wide range of injuries and disorders in the field of regenerative medicine and tissue engineering. In particular, MSCs have inherent characteristics to suppress the immune system and are being studied in clinical studies to prevent graft-versus-host disease. MSCs can be expanded in vitro and have potential for differentiation into multiple cell lineages. However, the impact of cell passaging on gene expression and function of the cells has not been determined.MethodsCommercially available human MSCs derived from bone marrow from six different donors, grown under identical culture conditions and harvested at cell passages 3, 5, and 7, were analyzed with gene-expression profiling by using microarray technology.ResultsThe phenotype of these cells did not change as reported previously; however, a statistical analysis revealed a set of 78 significant genes that were distinguishable in expression between passages 3 and 7. None of these significant genes corresponded to the markers established by the International Society for Cellular Therapy (ISCT) for MSC identification. When the significant gene lists were analyzed through pathway analysis, these genes were involved in the top-scoring networks of cellular growth and proliferation and cellular development. A meta-analysis of the literature for significant genes revealed that the MSCs seem to be undergoing differentiation into a senescent cell type when cultured extensively. Consistent with the differences in gene expression at passage 3 and 7, MSCs exhibited a significantly greater potential for cell division at passage 3 in comparison to passage 7.ConclusionsOur results identified specific gene markers that distinguish aging MSCs grown in cell culture. Confirmatory studies are needed to correlate these molecular markers with biologic attributes that may facilitate the development of assays to test the quality of MSCs before clinical use.

The ratio of matrix metalloproteinases (MMPs) to the tissue inhibitors of metalloproteinases (TIMPs) in wounded tissues strictly control the protease activity of MMPs, and therefore regulate the progress of wound closure, tissue regeneration and scar formation. Some amphibians (i.e. axolotl/newt) demonstrate complete regeneration of missing or wounded digits and even limbs; MMPs play a critical role during amphibian regeneration. Conversely, mammalian wound healing re-establishes tissue integrity, but at the expense of scar tissue formation. The differences between amphibian regeneration and mammalian wound healing can be attributed to the greater ratio of MMPs to TIMPs in amphibian tissue. Previous studies have demonstrated the ability of MMP1 to effectively promote skeletal muscle regeneration by favoring extracellular matrix (ECM) remodeling to enhance cell proliferation and migration. In this study, MMP1 was administered to the digits amputated at the mid-second phalanx of adult mice to observe its effect on digit regeneration. Results indicated that the regeneration of soft tissue and the rate of wound closure were significantly improved by MMP1 administration, but the elongation of the skeletal tissue was insignificantly affected. During digit regeneration, more mutipotent progenitor cells, capillary vasculature and neuromuscular-related tissues were observed in MMP1 treated tissues; moreover, there was less fibrotic tissue formed in treated digits. In summary, MMP1 was found to be effective in promoting wound healing in amputated digits of adult mice.