Isolation, selection and culture methods to enhance clonogenicity of mouse bone marrow derived mesenchymal stromal cell precursors

Claas Baustian, Shirley Hanley, Rhodri Ceredig

Conventionally cultured mouse bone marrow mesenchymal stromal cells (mBM-MSC) are a heterogeneous population that often initially contain contaminating haematopoietic cells. Variability in isolation methods, culture protocols and the lack of specific mBM MSC markers might explain this heterogeneity. The aim of this study is to optimise the isolation, culture conditions and selection of mBM-MSC. Mouse BM-MSCs were isolated from crushed long bones (cBM) or flushed bone marrow (fBM) from 6-8 week old C57Bl/6 mice. These subpopulations were analysed by flow cytometry using commonly used mBM-MSC cell surface marker, e.g. Sca-1, CD29 and CD44. Cells were cultured and expanded in vitro in hypoxic conditions of either 2 % or 5 % oxygen. Cell sorting and qRT-PCR was used to determine transcript levels of stem cell and lineage related genes in individual subpopulations. During early passaging not only do contaminating haematopoietic cells disappear, but there is a change in the phenotype of mBM-MSC affecting particularly CD44 and Sca-1 expression. By fluorescence activated cell sorting of CD45(-)/Ter119(-) mBM stroma based on Sca-1 expression and expansion in hypoxic conditions, we show that Sca-1(+) cells had higher CFU-F frequencies and showed enhanced proliferation compared with Sca-1(-) cells. As evaluated by in vitro assays and qRT-PCR, these cells presented in vitro tri-lineage differentiation along osteocyte, chondrocyte, and adipocyte lineages. Finally, by prospective isolation of Sca-1(+)PDGFRα(+)CD90(+) cells we have isolated mBM-MSC on a single cell level, achieving a CFU-F frequency of 1/4. Functional investigations demonstrated that these MSC clones inhibited T-lymphocyte proliferation. By positive selection using a combination of antibodies to Sca-1, CD90 and PDGFRα and culturing in hypoxia, we have found a subpopulation of BM cells from C57Bl/6 mice with a CFU-F cloning efficiency of 1/4. To our knowledge these results represent the highest frequencies of mouse MSC cloning from C57Bl/6 mice yet reported.