Miao_2004_Tissue.Eng_10_807

Bone marrow stromal cells (BMSCs) possess certain stem celllike properties and can differentiate to adopt a number of mesenchymal phenotypes. BMSCs are usually investigated in vitro as homogeneous single-cell suspensions; however, these preparations lose much of their osteogenic capacity. Using the fibroblastic colony-forming unit assay, we have compared the proliferation and capacity to express alkaline phosphatase of BMSC-containing aggregates of bone marrow cells with single-cell suspensions of bone marrow cells from the same source. Aggregates were separated from single cells by density gradient centrifugation or cell sieving. The aggregate and single-cell preparations gave rise to similar numbers of colonies; however, the colonies produced by the aggregates were larger and expressed higher levels of alkaline phosphatase. When the aggregates were dissociated, colonies still formed; however, they expressed negligible levels of alkaline phosphatase. Immunomagnetic selection and immunofluorescent staining for CD61, N-methyl-D-aspartate (NMDA) receptor subunit 1, and acetylcholinesterase showed that the majority of the aggregates giving rise to osteoblastic colonies contained megakaryocytes. These data demonstrate that removing BMSCs from their normal environment reduces their osteoblastic capacity and that to achieve their maximal differentiation, BMSCs require direct physical contact with accessory cells such as megakaryocytes. These findings may be of direct relevance to the use of BMSCs for tissue-engineering purposes.