| Mesenchymal stem cells (MSCs) are defined by their ability to self-renew and differentiate into multiple cell types, thus offering great promise for their use in tissue engineering and regenerative medicine. Currently, an increased effort to expand the therapeutic potential of MSCs has been limited by the inability to reproducibly characterize and isolate a reliable efficacious and homogeneous population. The main reason for variability is that hMSCs cultures contain a heterogeneous population of cells consisting of maximally multipotent cells and committed or senescent cells that are limited in plasticity. The proportion of multipotent cells varies as a result of tissue source, plating density and incubation time of each passage. In this study, we focused on identifying protein markers that may be employed to predict the efficacy of a cultured MSC population. Markers of progenitor status were identified by comparison of MSCs at early and late passage, donor-matched skin fibroblasts, and commercially available dermal fibroblast cell lines. Differentiation potential was determined by in vitro assays of osteogenesis, adipogenesis and chondrogenesis. Low passage MSCs differentiated into all three lineages, while late passage MSCs and both fibroblast preparations did not. To identify novel markers of early progenitors, microarray transcript analysis between early passage MSCs and fibroblasts was performed. Messenger RNA encoding the cytokine leukemia inhibitory factor (LIF) was identified as differentially expressed. ELISA on conditioned media confirmed that LIF secretion was much higher from early progenitor MSCs than donor matched or commercial lines of fibroblasts and dropped with extensive expansion or induction of differentiation. In clonally expanded MSCs, colonies that retained progenitor status expressed significantly higher levels of LIF than those that failed to differentiate. We found that the addition of LIF to MSC cultures during expansion resulted in a small increase in the number of colony-forming units, however did not maintain the progenitor status of the MSCs. The addition of LIF activated JAK/STAT signaling in low and high passage MSC cultures. Our results indicate LIF expression may represent a marker to quantify the differentiation potential of MSCs and may be especially suited for the rapid non-invasive quality control of clinical preparations. |
