Differential Expression of Neuron-Glial Antigen 2 (NG2) Selects for Cell Survival during High-Throughput Enrichment of Marrow-Derived Mesenchymal Stem Cells

Cellular heterogeneity of mesenchymal stem cells (MSCs) impedes their use in regenerative medicine.  The objective of this research is to identify potential biomarkers for the high-throughput enrichment of progenitors from heterogeneous MSC cultures.  To this end, the present study examines variation in expression of neuron-glial antigen 2 (NG2) and melanoma cell adhesion molecule (CD146) on the surface of MSCs derived from human bone marrow in response to culture conditions and among cell populations.  Multipotent cells isolated from heterogeneous MSC cultures exhibit a >3-fold increase in surface expression for NG2 and >2-fold increase for CD146 as compared with parental and lineage-committed MSCs.  For both antigens, surface expression is downregulated by ≥6-fold when MSCs become confluent.  During serial passage, maximum surface expression of NG2 and CD146 is associated with minimum doubling time.  Upregulation of NG2 and CD146 during loss of adipogenic potential at early passage suggests some limits to their utility as potency markers.  A potential relationship between proliferation and antigen expression was explored by sorting heterogeneous MSCs into rapidly and slowly dividing groups.  Fluorescence-activated cell sorting revealed that rapidly dividing MSCs display lower scatter and 50% higher NG2 surface expression than slowly dividing cells, but CD146 expression is comparable in both groups.  Heterogeneous MSCs were sorted based on scatter properties and surface expression of NG2 and CD146 into high (HI) and low (LO) groups.  Sc^LONG2^HI and Sc^LONG2^HICD146^HI MSCs have the highest proliferative potential of the sorted groups, with colony-forming efficiencies that are 1.5-2.2 times the value for the parental controls.  The Sc^LO gate enriches for rapidly dividing cells.  Addition of the NG2^HI gate selects for enhanced cell survival.  Further addition of the CD146^HI gate does not significantly improve cell division or survival.  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 the Sc^LONG2^HI sort strategy.  This research was funded by the National Science Foundation.