Culture and labeling of bone marrow mesenchymal stem cells from green fluorescence protein mice: A comparative study

Objective To obverse and compare the two isolation and culture methods for bone marrow mesenchymal stem cells（BMSCs） and study the feasibility to use green fluorescence protein（GFP）-BMSCs as a labeling method in transplantation experiment. Methods Proliferation of BMSCs cultured with compact bone culture method（A） and whole bone marrow culture method（B） was induced and other culture methods were used to optimize fluid-changing measures.Morphological changes in GFP-BMSCs cultured with methods A and B were observed.Activity of BMSCs in GFP mice was identified according to the growth curve and multi-differentiation functions of BMSCs after the third passage.The stability of BMSCs in GFP mice after several passages and green fluorescence-induced differentiation was observed. Results 1)BMSCs were proliferated into clone colonies with different sizes,and then further expanded around and integrated with the adjacent clone colonies after primary culture with method A.The cells after the third or forth passage and the purified BMSCs were obtained after primary culture with method B because they were mixed with many blood cells.2)The growth curve of BMSCs was observed after cultured with methods A and B.The BMSCs proliferated rapidly and their purity was high after cultured with method A.However,the BMSCs were difficult to proliferate due to the influence of other cells.3)The BMSCs with a higher activity cultured with methods A and B could be induced to adipocytes with oil O staining.The bone structure of some aggregated BMSCs was observed during osteogenesis differentiation.4)The GFP-BMSCs after several passages were characterized by stable biological properties and the GFP was strongly expressed after 5 passages and induction of differentiation. Conclusion More GFP-BMSCs with a high concentration can be obtained with bone slice culture method than with traditional method after primary or first passage.Their fluorescence is not diminished after several passages and can thus be used as a labeling method in vivo.