Isolation of mouse MSC is generally more difficult than human and rat MSC due to a high number of contaminating haematopoietic lineage cells and slow cell growth[2
]. Studies have also demonstrated mouse strain-dependent variability of MSC in terms of growth kinetics, surface markers and potential for differentiation[16
]. With the protocol described here for bone marrow-derived MSC expansion, we provide descriptive data that MSC can be successfully generated from 3 different mouse strains, namely CBA/Ca, ICR and Balb/c. Bone marrow cells were cultured and each culture was characterised for their morphology, immunophenotype and differentiation potential. MSC from all strains tested showed expression of relevant surface markers and differentiation potential into osteogenic and adipocytic cells, albeit some differences in terms of time to achieve MSC immunophenotype.
Isolation of MSC from mouse bone marrow usually results in a highly heterogenous cell population with a high degree of haematopoietic contaminants such as macrophages and fibroblasts[2
]. Immunophenotyping analysis revealed early passages of our bone marrow cultures to be positive for haematopoietic markers CD45 and CD11b (a macrophage-specific marker). Continuous change of cell culture medium removes non-adherent haematopoietic populations from the cultures[3
]. Passaging bone marrow cultures also helps eliminate haematopoietic contaminants and yield purer MSC cultures[17
]. With these approaches, we accordingly observed the MSC cultures become increasingly homogenous at later passages with minimal CD45 expression profile by P7-P8, although it took a little longer for cells from Balb/c mice. Better quality MSC cultures were also obtained by discarding cells that remained attached to flasks following trypsinisation. These firmly adherent cells are probably contaminating fibroblasts and have been reported by other laboratories[17
]. Expression of stem cell marker Sca-1 also increased at later passages. Sca-1 is a marker associated with MSC and also haematopoietic and endothelial progenitors[20
]. MSC also expressed positivity to CD44 and CD106 and negativity to MHC II. To further encourage MSC growth and colony formation, we used 15% of a mouse mesenchymal supplement (MesenCult®
, STEMCELL Technologies) instead of the routine 10% FBS to obtain enriched MSC cultures.
In addition to morphology and phenotype, the characterisation of MSC is complete with demonstration of their capacity for multilineage mesenchymal differentiation[15
]. All 3 mouse strains exhibited multilineage potential in vitro
, differentiating into adipocytes and osteocyte when stimulated with appropriate induction media. MSC from Balb/c mice more readily differentiate into adipocytes than the other strains, also shown by Peister and colleagues who found Balb/c more readily differentiating into adipose cells than C57Bl/6J and DBA 1 mice[17
]. Conversely, MSC from Balb/c took longer to differentiate into osteocytes compared to the other 2 strains tested in our study. In view of the duration that MSC cultures remained in suitable phenotype and their ease to differentiation, our group has primarily utilised ICR mice for downstream experimentation where we show these stem cells to have immunomodulatory properties[13
MSC from all 3 different strains of mice tested were suitable sources for bone marrow-derived MSC as they showed typical morphology, immunophenotype and differentiation capacities of MSC. However, bone marrow cultures from Balb/c mice took longer than the other strains to achieve MSC immunophenotype.