Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow

Cultures of plastic-adherent cells from bone marrow have attracted interest because of their ability to support growth of hematopoietic stem cells, th eir multipotentiality for differentiation, and their possible use for cell and gene therapy. Here we found that the cells grew most rapidly when they were initially plated at low densities (1.5 or 3.0 cells/cm(2)) to generate single-cell derived colonies. The cultures displayed a lag phase of about 5 days, a log phase of rapid growth of about 5 days, and then a stationary phase. FAGS analysis demonstrated that stationary cultures contained a majo r population of large and moderately granular cells and a minor population of small and agranular cells here referred to as recycling stem cells or RS -1 cells. During the lag phase, the RS-1 cells gave rise to a new populatio n of small and densely granular cells (RS-2 cells). During the late log pha se, the RS-2 cells decreased in number and regenerated the pool of RS-1 cel ls found in stationary cultures. In repeated passages in which the cells we re plated at law density, they were amplified about 10(9)-fold in 6 wk. The cells retained their ability to generate single-cell derived colonies and therefore apparently retained their multipotentiality for differentiation.