Isolation, expansion and characterization of single mesenchymal stem cells

Mesenchymal Stem Cells (MSCs) are multipotent precursors to many mesodermal cell lineages in vertebrate animals and are most often obtained from bone marrow. Certain attributes of MSCs, including migration toward sites of inflammation, ease of transduction, and lack of immunogenicity, suggest these cells may be potentially useful for regenerative medicine. Putative therapeutic uses include regeneration of damaged tissue, acting as a vessel for delivering a therapeutic transgene, support of other cell types for tissue repair, and modulating the immune reaction to co-transplanted cells or tissues. However, MSCs lack distinctive surface markers and conventional MSC culture has been shown to be heterogeneous. A thorough characterization of MSC culture at a single cell level has not been adequately demonstrated. These conditions lead to the question of whether there are true mesenchymal "stem" cells, or simply a mixed population of committed mesenchymal progenitors. In addition to being an important question in MSC biology, this may prove to be an important distinction in certain therapeutic settings. In this study, the methods used to identify hematopoietic high proliferative potential-colony forming cells (HPP-CFCs) and high proliferative potential- endothelial colony forming cells (HPP-ECFCs), were adapted to investigate the existence of high proliferative potential-mesenchymal colony forming cells (HPP-MCFCs), and the differentiation potential of these cells toward adipogenic, chondrogenic, and osteogenic lineages at a single cell level. This study demonstrates for the first time that a hierarchy of mesenchymal cells within conventional MSC culture can be described, and single HPP-MCFCs can differentiate toward adipogenic, chondrogenic and osteogenic lineages as well as form secondary colonies.