Human mesenchymal stem cells or multipotent stromal cells (MSCs) are of interest for clinical therapy, in part because of their capacities for proliferation and differentiation. However, results from clinical trials and in vitro models have been variable, possibly due to MSC heterogeneity and a lack of standardization between MSC in vitro expansion protocols. Here, we defined changes in MSCs during expansion in vitro. We assayed cultures of human MSCs from multiple donors, plating densities, times between feeding, percentage confluence, and passages. Differences in the MSCs in vitro were then assayed in vivo for engraftment in normal hippocampi and hearts after infarction.

In culture, we determined the MSCs undergo transition from division to development. We found the molecular variations between different donors were less than the variation within the expansion of MSCs from a single donor at different time points in culture. Similarly, the variation between cells from different passages was found to be less than the differences between cells at early and late time points of the same passage. This general trend for the MSCs to transition during culture could be delayed when cells were fed with greater frequency, especially evident in higher density cultures. This manipulation was able to maintain MSCs in a more division focused milieu and conversely delay early markers of differentiation, accompanying quiescence. This transition was highly dependent on time in culture, specifically after replating. We were also able to better characterize MSC plasticity when reset by replating at the molecular and gross morphological levels. This plastic ability decreased with passage until eventual senescence. The results demonstrated major differences in cultures of MSCs that should be considered when using the cells in laboratory and clinical applications.