Non-multipotent stroma inhibit the proliferation and differentiation of mesenchymal stromal cells in vitro

Background aims. The ability to expand and maintain bone marrow (BM)-derived mesenchymal stem cells (MSC) in vitro is an important aspect of their therapeutic potential. Despite this, the exact composition of stromal cell types within these cultures and the potential effects of non-stem cells on the maintenance of MSC are poorly understood. Methods. C57BL/6J BM stroma was investigated as a model to determine the relationship between MSC and non-multipotent cells in vitro. Whole BM and single-cell derived cultures were characterized using flow cytometry and cell sorting combined with multipotent differentiation. Proliferation of individual stromal populations was evaluated using BrdU. Results. At a single-cell level, MSC were distinguished from committed progenitors, and cells lacking differentiation ability, by the expression of CD105 (CD105<SU+</SU). A 3-fold reduction in the percentage of CD105<SU+</SU cells was detected after prolonged culture and correlated with loss of MSC. Depletion of CD105<SU+</SU cells coincided with a 10-20% increase in the frequency of proliferating CD105<SU-</SU cells. Removal of CD105<SU-</SU stroma caused increased proliferation in CD105<SU+</SU cells, which could be diminished by conditioned media from parent cultures. Comparison of the multipotent differentiation potential in purified and non-purified CD105<SU+</SU cells determined that MSC were detectable for at least 3 weeks longer when cultured in the absence of CD105<SU-</SU cells. Conclusions. This work identifies a simple model for characterizing the different cellular components present in BM stromal cultures and demonstrates that stromal cells lacking multipotent differentiating capacity greatly reduce the longevity of MSC.