Differentiation of rabbit bone marrow mesenchymal stem cells labeled by green fluorescent protein into osteoblasts and lipoblasts

Wenjian CHEN; Feng LI; Song ZHOU; Anmin CHEN

ABSTRACT

BACKGROUND:

Labeling was commonly used to further trace bone marrow mesenchymal stem cells (BMSCs).

OBJECTIVE:

To label rabbit BMSCs by green fluorescent protein,and then to observe the potency of multi-directional differentiation by osteoblast and lipoblast formation induction.DESIGN,TIME AND

SETTING:

Cell observational study was performed at the Laboratory of the Department of Orthopaedics,Tongji Hospital Affiliated to Tongji Medical College,Huazhong University of Science and Technology from December 2007 to October 2008.MATERIALSTotally 6 healthy New Zealand rabbits aged 4 months were purchased from the Animal Experimental Center,Tongji Medical College,Huazhong University of Science and Technology.

METHODS:

BMSCs were isolated from the femurs of rabbits,purified and cultured in vitro by density gradient centrifugation and adherent culture.BMSCs at the third passage were obtained.Green fluorescent protein plasmid was transfected into BMSCs using Lipofectamine.The stable transfection cells were obtained using G418 selection after transfection.The stable transfected BMSCs were induced to osteoblasts and lipoblasts by osteogenic inductor and adipogenic inductor.MAIN OUTCOME

MEASURES:

The following parameters were measuredBMSC morphology,surface marker expression,green fluorescent protein labeling,the differentiation potential of BMSCs into osteoblasts and lipoblasts.

RESULTS:

The BMSCs were mostly fusiform in shape,to be similar to fibroblast cell after cultivation of primary and subculture.The flow cytometer analysis showed that the membrane mark CD44 was positive,CD34 was negative.Following G418 screening,BMSCs labeled by green fluorescent protein were apparent calcium mineralization after 21 days osteogenic induction.Alizarin red staining showed positive with red.Intra-cellular lipid droplet appeared after 3 days adipogenic induction.2 weeks later,oil red O staining showed a great quantity lipid deposition.

CONCLUSION:

Rabbit BMSCs labeled by green fluorescent protein using Upofectamine have the potency of multi-directional differentiation after induction.