Prospective isolation and characterisation of mouse bone marrow-derived mesenchymal stromal cells

Abstract

Conventionally cultured mouse bone marrow (mBM) mesenchymal stromal cells (MSC) are a heterogeneous population that often initially contain contaminating haematopoietic cells. Variability in isolation methods, culture protocols and the lack of specific MSC surface markers might explain this heterogeneity. In this thesis it is shown that during early passaging of bone chip-derived MSC not only do haematopoietic cells disappear, but there is also a change in surface marker expression. To further dissect bulk MSC populations, fluorescence activated cell sorting of mBM suspensions based on Sca-1 expression among non-hematopoietic cells was carried out and cells expanded in hypoxic conditions. During early passaging, there was a change in the surface phenotype of MSC affecting particularly CD44 and Sca-1 expression. It became evident that CFU-F frequencies and proliferation was greater among Sca-1+ compared with Sca-1- cells. As evaluated by in vitro differentiation and qRT-PCR assays, both populations were capable of tri-lineage differentiation along osteocyte, chondrocyte, and adipocyte lineages. By prospective isolation of Sca-1+PDGFRalpha+CD90+ non-hematopoietic mBM cells, clones of MSC could be isolated with a CFU-F frequency of 1/4. This is one of the highest CFU-F frequencies for mouse MSCS reported so far. Functional investigations demonstrated that these MSC clones had immuno-modulatory activity in that they inhibited T-lymphocyte proliferation.