Gene expression analysis at the single cell level using the human bone marrow stromal cell as a model: sample preparation methods.

Recent advances in molecular technology, including gene expression microarray analysis, have allowed researchers to examine global patterns of gene expression at high resolution in populations of cultured cells or tissues. Although these techniques can be applied with great sophistication and are useful for address ing many biological questions in cell populations, it is also of great value to assess gene expression at the level of the single cell. This can be achieved by one of two different approaches: (1) specific cell types can be purified from heterogeneous tissues or cultures using immunological methods such as fluorescence-based or magnetic cell sorting or laser capture microdissection, followed by amplification of target cell nucleic acids, and analysis of expressed genes; or (2) immunohisto-chemical studies and in situ expression studies on identical tissue sections can be used to identify genes or sets of genes whose expression correlates with a morpho logically or immunochemically distinct cell-type. Using either approach, the target cell types are identified by their morphological or immunohistochemical properties. This chapter is a primer on using single cell gene expression technology to study human bone marrow stromal cells that express mixed lineage markers. Cytomorphological, cytochemical, and immunocytochemical methods as well as gene expression microarray studies demonstrated that single stromal cells simulta neously express markers associated with osteoblast, fibroblast, muscle, and adi-pocyte differentiation, suggesting that these stromal cells are mesenchymal progenitor cells that have multilineage differentiation capacity. These data charac terize human bone marrow stromal cells as adult stem cells. Because of their pluripotent nature, single cell gene expression technology is particularly critical for characterizing and developing the therapeutic potential of these cells.