【Objective】 Mesenchymal stem cells (MSC) are multipotential stromal cells. Now the MSC of human and many animals have been isolated and identified successfully. However

the isolation of purified murine MSC from bone marrow is far more difficult than that in human and other species

so the clonal analysis results of murine bone marrow MSC are limited and even inconsistent. Therefore

isolation of pure murine MSC in vitro and identification of the biological characteristics of colony-forming units(CFU)were further studied. 【Methods】 Mononuclear cells flushed from thigh bone marrow cavity of C57BL/6 mouse were plated at low density. Three types of CFU with different morphology and proliferation characteristics were acquired by limiting dilution and cloning selection. The immunophenotype of these three types of CFU were analyzed by flow cytometry and the osteogenic differentiation and adipocytic differentiation potential were identified by Alizarin red staining and Oil Red O staining. 【Results】 Thus

by low density culture system combined with cloning isolation technique

we totally obtained three distinct clonal MSC populations from C57BL/6 mouse bone marrow. These cell lines exhibited different morphology: MSC1 had a near round morphology

while MSC2 presented a typical fibroblast-like morphology and MSC3 had fusiform and polygonal morphology. These three cell populations were all strong positive for stem cell antigen-1 (Sca-1)

negative for CD11b and CD45 and partially positive for CD90.2. Differentiation potential assay showed that MCS1 could only be induced to adipocyte and MSC2 only to osteoblast

while MSC3 could differentiate to adipocyte and osteoblast.【Conclusions】 Low density culture system combined with cloning isolation technique could isolate mouse mesenchymal stem cells successfully. Mouse MSC are highly heterogeneous cell populations which may contain multi-potential MSC or uni-potential progenitors at different developmental stages.