(84g) Resolving Proliferation-Potency Relationship Identifies Survival Marker for Multipotent Mesenchymal Stem Cells
Heterogeneity in cell survival is a major impediment to realizing the therapeutic potential of mesenchymal stem cells (MSCs). The present study resolves the proliferation-potency relationship for MSCs at the clonal level and employs this knowledge to identify a novel cell-surface marker to enrich multipotent MSCs with enhanced survival. Clonal analysis of the proliferation-potency relationship was performed with a unique high-capacity assay that generates single-cell derived MSC colonies that are classified according to their trilineage potential to exhibit adipo- (A), chondro- (C), and osteogenesis (O) as a measure of potency. The proliferation potential of MSC clones was analyzed from complementary approaches of colony-forming efficiency, ex vivo expansion, senescence and cell death. These different approaches yielded similar results, which makes a compelling argument for the following proliferation-potency relationship that was detected. There was a broad range of proliferation potentials of clones isolated from early passage MSCs. For example, the median specific growth rate was 0.85 per day (20 h doubling time) for OAC clones and was 5-fold less for O clones (P<0.01). As multipotent MSCs progressed through a complex hierarchy of lineage commitment, proliferation potential correlated to overall potency, but it was independent of the specific lineage pathway of commitment in that OA and OC clones had similar proliferation potentials. The gradient in this correlation was pronounced with highly clonogenic OAC clones at one extreme and senescent O clones at the other. In fact, more than 75% of cells in O clonal cultures stained positive for senescence-associated beta-galactosidase activity vs. less than 10% for OAC clones (P<0.001). The proliferation-potency relationship served as a template to identify neuron-glial antigen 2 (NG2) as a cell-surface marker for MSC survival. Fluorescence-activated cell sorting revealed that MSCs selected for low scatter and high NG2 surface expression had a 50% improvement in clonogenic survival in vitro relative to low scatter MSCs and a 100% improvement relative to parental MSCs. This is the first report of a role for NG2 in the survival of normal diploid cells. The relationship between NG2 and MSC survival is particularly significant because cell survival is a basic prerequisite for any MSC therapy to be effective. As such, there are numerous basic research and clinical applications for NG2as a MSC survival marker. This research was funded by NIH (R03EB007281) and NSF (CBET-1066167).