Onset of heterogeneity in culture-expanded bone marrow stromal cells

Inconsistencies among in vitro and in vivo experiments using adult mesenchymal stem cells (MSCs) confound development of therapeutic, regenerative medicine applications, and in vitro expansion is typically required to achieve sufficient cell numbers for basic research or clinical trials. Though heterogeneity in both morphology and differentiation capacity of culture-expanded cells is noted, sources and consequences are not well understood. Here, we endeavored to observe the onset of population heterogeneity by conducting long-term continuous in vitro observation of human adult bone marrow stromal cell (BMSC) populations, a subset of which has been shown to be stem cells (also known as bone marrow-derived MSCs). Semi-automated identification and tracking of cell division and migration enabled construction of cell lineage maps that incorporated cell morphology. We found that all BMSCs steadily grew larger over time; this growth was interrupted only when a cell divided, producing two equally sized, morphologically similar daughter cells. However, a finite probability existed that one or both of these daughters then continued to increase in size without dividing, apparently exiting the cell cycle. Thus, larger BMSCs are those cells that have exited the normal cell cycle. These results hold important implications for MSC in vitro culture expansion and biophysical sorting strategies.