Purification, Expansion, and Flow Cytometry-Based Phenotyping of Mouse Derived Bone Marrow Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of stem cells that can self-renew and differentiate into multiple cell lineages. Among them, MSCs found in bone marrow, known as bone marrow mesenchymal stem cells (BMSCs), play a crucial role in osteogenesis, hematopoietic support, homing/migration, and immunosuppression. BMSCs are widely used in cell biology and tissue engineering research due to their potential for cellular therapeutic strategies, as well as their easy availability, genetic stability, and low immunogenicity. Hematopoietic stem cells (HSCs) and BMSCs coexist in bone marrow but have distinct progenitor cells. When extracting cells from bone marrow, multiple cell types are typically obtained, which can lead to heterogeneity in the cell population, potentially causing bias and not meeting high experimental standards. Therefore, it is crucial to develop effective techniques to improve the homogeneity and yield of BMSCs in vitro. This study presents a detailed protocol for the purification, expansion, and flow cytometry-based phenotyping of BMSCs obtained from BALB/c mice. The homogeneity and stem cell phenotype of third-passage BMSCs were characterized using flow cytometry, demonstrating positivity for CD29, CD44, and Sca-1, negativity for CD45, and CD31, and confirming their osteogenic and adipogenic differentiation ability. This cost-effective protocol provides an easy and efficient method to obtain a sufficient number of homogeneous BMSCs with high proliferation and differentiation potential, which is essential for advancing research in mouse models and extensive preclinical studies.