Human adult stem cells (hMSC) from bone marrow stroma: Characterization and standardization of culture preparation for in vivo applications, and a possible new application ofhMSCs as a model system for differentiation
Description
For in vivo and clinical applications of human adult stem cells from bone marrow stroma (hMSCs), culture preparation standards are needed to ensure that preparations of cells are suitable for use. During previous work carried out to characterize hMSCs, a colony-forming unit (CFU) assay was used to demonstrate that the clonal efficiency of culture preparations correlated with their multi-potentiality. Additionally, CFU assays were useful for optimizing the culture conditions for expanding hMSCs because the conditions that preserved the %CFUs, could be identified and adopted relatively quickly. However, the CFU assay is inherently retrospective in scope. It requires 2 weeks to get the result and determine if a sample has a high enough CFU to meet quality standards. In clinical applications the outcome of quality assessment might need to be known for the very preparation that is to be used just before it is administered. For these reasons a flow cytometric measurement was developed that can predict which cell samples will have higher CFU efficiency and therefore greater multi-potentiality Additionally, an analysis of gene expression was carried out to identify genes that were co-expressed during the time course of differentiation of hMSCs into chondrocytes or adipocytes using three different differentiation protocols. Co-expressed genes were examined to identify new target genes for transcription factors that drive differentiation of hMSCs. Hierarchical clustering of differentially expressed genes was carried out to define groups of similarly behaving genes within each experiment. Next, groups of genes that were co-expressed in all three differentiation experiments were defined. The data were used to identify new targets of transcription factors involved in the differentiation of human cells into adipocytes and chondrocytes by introducing a proportionality criterion into our definition of co-regulation. Co-regulation was defined more stringently by requiring a highly significant linear regression between the levels of expression of known targets of transcription factors that drive chondrogenesis or adipogenesis and additional co-expressed genes not previously shown to be targets of the same transcription factors. Using this approach we identified 91 genes that are candidate downstream targets for SOX9, SOX5, C/BEP-alpha or PPAR-gamma