Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate

Marrow stromal cells (MSCs) were isolated from bone marrow obtained by aspi rates of the iliac crest of normal volunteers, The cells were isolated by t heir adherence to plastic and then passed in culture, Some of the samples e xpanded through over 15 cell doublings from the time frozen stocks were pre pared. Others ceased replicating after about four cell doublings. The repli cative potential of the cells in culture was best predicted by a simple col ony-forming assay in which samples from early passages were plated at low d ensities of about 10 cells per cm(2). Samples with high colony-forming effi ciency exhibited the greatest replicative potential. The colonies obtained by plating early passage cells at low density varied in size and morphology . The large colonies readily differentiated into osteoblasts and adipocytes when incubated in the appropriate medium. As samples were expanded in cult ure and approached senescence, they retained their ability to differentiate into osteoblasts. However, the cells failed to differentiate into adipocyt es. The loss of multipotentiality following serial passage in culture may h ave important implications for the use of expanded MSCs for cell and gene t herapy.